We recently performed studies to reduce the size of Inf2 to determine whether additional candidate genes emerged, and identified MHC class II genes as important determinants of susceptibility. We also previously identified by positional cloning the toll-like receptor 4 (Tlr4) as a candidate gene responsible for O3-induced pulmonary hyperpermeability and inflammation. The objective of the present study was to utilize transcriptomics to determine the mechanism by which TLR4-mediates O3-induced lung inflammation and injury. C3H/HeJ (HeJ;Tlr4 mutant), C3H/HeOuJ (OuJ;Tlr4 sufficient), Hspa1a/Hspa1btm1Dix (C57BL/6 background, Hsp70-/-) and C57BL/6 (Hsp70+/+) mice were used for these studies. Mice were exposed continuously to 0.3 ppm O3 or filtered air for 6, 24, 48 or 72 hr. Affymetrix Mouse430A_MOE gene arrays were used to analyze lung homogenates from HeJ and OuJ mice followed by GeneSpring analysis and PCR confirmation. Inflammation was assessed by bronchoalveolar lavage of the right lung and molecular analysis (ELISA and transcription factor activity) was performed on the left lung. Genes were identified with 2-fold changes with significant interactions (p<0.05) for strain and time in OuJ versus HeJ mice. PCR confirmed up-regulation of several genes identified in OuJ Hspa1b (Hsp70), Hspcb, Dnaja1;24-48 h and HeJ (Marco, Eln, and Saa3;24-48 h) mice. O3-induced expression of HSP70 protein was increased in OuJ compared to HeJ mice following 24-48 h O3. We then used Hsp70-/- mice to determine if HSP70 had a functional role in O3-induced inflammation. Polymorphonuclear leukocyte infiltration and lung hyperpermeability were significantly reduced in response to 48 h continuous O3 in the Hsp70-/- compared to Hsp70+/+ mice (p<0.05). MIP2 protein content and phospho-c-jun activity were also significantly reduced after 6 h O3 exposure in Hsp70-/- compared to Hsp70+/+ mice (p<0.05). These studies suggest that HSP70 is involved in the regulation of O3-induced lung inflammation likely through the TLR4 pathway. Previous studies have shown that expression of chemokines and their receptors is elevated in human inflammatory processes. Enhanced expression of the chemokine receptor Cx3cr1 results in enhanced leukocyte adhesion. Cx3cr1 is known to play a major role in chronic pulmonary inflammation;however very little is known concerning their role in response to sub-acute pulmonary injury. Interleukin-17 (IL-17) is linked to neutrophilic mobilization in chronic pulmonary inflammation. Previous research suggests that IL-17 is not expressed in Cx3cr1-/- mice, suggesting that IL-17 and Cx3cr1 work together in the inflammatory response. The purpose of this research was to determine the role of Cx3cr1 in O3-induced pulmonary inflammation, and the role IL-17 plays in this response. Cx3cr1+/-, Cx3cr1-/-, Il17r+/+ and Il17r-/- mice were exposed to air or 0.3 ppm O3 for 24, 48 or 72 hours. Bronchoalveolar lavage (BAL) fluid was collected after exposure and analyzed for total protein (a marker of lung permeability), the numbers of inflammatory cells (macrophage, neutrophils, and lymphocytes) and indicators of pulmonary injury (epithelial cells). Pulmonary PCNA levels were measured by western blot analysis, and immunohistochemistry. Cytokine IL-17A expression in lung homogenate was analyzed by ELISA. Relative to respective air controls, 48 and 72 hr exposure to O3 caused a significant increase in BALF total protein and numbers of inflammatory cells in all mice. However, significantly greater BALF protein content and numbers of inflammatory cells were found in O3-exposed Cx3cr1+/- mice compared to Cx3cr1-/- mice. Slight elevation of IL-17R was detected in Cx3cr1+/- mice after exposure to O3, but IL-17R was not detected in Cx3cr1-/- mice after O3 exposure. Significantly greater numbers of neutrophils were found in Il17ra+/+ mice compared to Il17ra-/- mice after exposure to ozone for 48 hours. O3-induced inflammatory cell infiltration, change in lung permeability, and IL-17R expression was significantly diminished in Cx3cr1-/- mice relative to Cx3cr1+/- mice. This suggests that Cx3cr1 contributes to the pulmonary inflammatory response to O3 exposure, and it appears that IL-17 does play a role in this inflammatory response. Children may be more at risk to air pollution than adults due to higher minute ventilation rates and activity levels outdoors, and continued lung development into adolescence. To study the impact of O3 exposure on the developing lung, we have collaborated with Dr. Edward Postlethwait (Univ Alabama, Birmingham) to study O3 effects on gene expression in infant rhesus macaque monkeys that were exposed to a regimen mimicking urban conditions and site and exposure duration samples were obtained for gene expression analysis. Primates were raised in filtered air (FA) and nighttime exposures to 0.5 ppm O3 conducted for 1 cycle (9 d FA followed by 8 hrs/d O3 for 5 d), 11 cycles, or FA. Exposures ended at 180 d of age. Immediately post exposure, lungs were microdissected to obtain central axial airways (generation 8-10) and terminal bronchioles devoid of parenchyma, and stored in RNAlater. RNA was pooled to provide a single sample for each experimental group. Gene expression (Agilent rhesus monkey oligo microarrays) was analyzed initially by K-means clustering. Informative patterns were analyzed (Ingenuity) to identify interaction between differentially expressed genes. A number of informative patterns were identified. 1) Genes upregulated in axial and terminal bronchiole tissue after 1 cycle: inflammatory and immune responses (e.g. IL8, TNF). 2) Upregulated genes in axial tissue after 11 cycles: cellular inflammation and hematological system (IL1B, IL17, MAPK). 3) Genes differentially expressed in axial and terminal bronchioles irrespective of O3 exposure: developmental (ACAN, MUC2, CX3CL1) and immune function (IL17RD, FGF1, DAP1). In infant primates, the superimposition of injury and repair on growth and development results in anatomic and exposure specific alterations in gene expression that is likely coupled to the O3-induced structural, inflammatory, and biochemical effects also observed. The Glutathione-S-Transferase Mu 1 null genotype has been reported to be a risk factor for acute respiratory disease associated with increases in ambient air O3. However, it is not known if GSTM1 modulates these O3 responses in vivo in humans. The purpose of this study was to determine if the GSTM1 null genotype modulates O3 responses in humans. Thirty-five normal volunteers were genotyped for the GSTM1 null mutation and underwent a standard O3 exposure protocol to determine if lung function and inflammatory responses to O3 were different between the 19 GSTM1 normal and 16 GSTM1 null volunteers. GSTM1 did not modulate lung function responses to acute O3. Granulocyte influx 4 hours after challenge was similar between GSTM1 normal and null volunteers. However, GSTM1 null volunteers had significantly increased airway neutrophils 24 hours after challenge, as well as increased expression of HLA-DR on airway macrophages and dendritic cells. The GSTM1 null genotype is associated with increased airways inflammation 24 hours following O3 exposure, consistent with the lag time observed between increased ambient air O3 exposure and exacerbations of lung disease. These observations suggest that the GSTM1 null genotype likely confers increased risk for exacerbation of O3-induced lung disease through promoting an enhanced neutrophilic and monocytic inflammatory response to O3.
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