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 cytokines and their receptors is elevated in human inflammatory processes. However, the mechanisms underlying ozone (O3)-induced pulmonary inflammation remain unclear. Interleukin (IL)-10 is an anti-inflammatory cytokine that is known to inhibit inflammatory mediators. Objectives: The current study investigated the molecular mechanisms underlying IL-10-mediated attenuation of O3-induced pulmonary inflammation in mice. Methods: Il10-deficient (Il10-/-) and wild type (Il10+/+) mice were exposed to 0.3-ppm O3 or filtered air for 24, 48 or 72 hr. Immediately following exposure, differential cell counts, and total protein (a marker of lung permeability) were assessed from bronchoalveolar lavage fluid (BALF). mRNA and protein levels of cellular mediators were determined from lung homogenates. We also utilized global mRNA expression analyses of lung tissue with Ingenuity Pathway Analyses (IPA) to identify patterns of gene expression through which IL-10 modifies O3-induced inflammation. Results: Mean numbers of BALF polymorphonuclear leukocytes (PMNs) were significantly greater in Il10-/- mice than in Il10+/+ mice after exposure to O3 at all time points tested. O3-enhanced nuclear NF-B translocation was elevated in the lungs of Il10-/- compared to Il10+/+ mice. Gene expression analyses revealed several IL-10 and O3-dependent mediators, including MIP-2, cathepsin E, and serum amyloid A 3. Conclusions: Results indicated that IL-10 protects against O3-induced pulmonary neutrophilic inflammation and cell proliferation. Moreover, gene expression analyses identified three response pathways and several genetic targets through which IL10 may modulate the innate and adaptive immune response. These novel mechanisms of protection against the pathogenesis of O3-induced pulmonary inflammation may also provide potential therapeutic targets to protect susceptible individuals. 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. Asthma is a known risk factor for acute ozone-associated respiratory disease. Ozone causes an immediate decrease in lung function and increased airway inflammation. The role of atopy and asthma in modulation of ozone-induced inflammation has not been determined. We sought to determine whether atopic status modulates ozone response phenotypes in human subjects. Fifty volunteers (25 healthy volunteers, 14 atopic nonasthmatic subjects, and 11 atopic asthmatic subjects not requiring maintenance therapy) underwent a 0.4-ppm ozone exposure protocol. Ozone response was determined based on changes in lung function and induced sputum composition, including airway inflammatory cell concentration, cell-surface markers, and cytokine and hyaluronic acid concentrations. All cohorts experienced similar decreases in lung function after ozone. Atopic and atopic asthmatic subjects had increased sputum neutrophil numbers and IL-8 levels after ozone exposure;values did not significantly change in healthy volunteers. After ozone exposure, atopic asthmatic subjects had significantly increased sputum IL-6 and IL-1beta levels and airway macrophage Toll-like receptor 4, FcepsilonRI, and CD23 expression;values in healthy volunteers and atopic nonasthmatic subjects showed no significant change. Atopic asthmatic subjects had significantly decreased IL-10 levels at baseline compared with healthy volunteers;IL-10 levels did not significantly change in any group with ozone. All groups had similar levels of hyaluronic acid at baseline, with increased levels after ozone exposure in atopic and atopic asthmatic subjects. Atopic asthmatic subjects have increased airway inflammatory responses to ozone. Increased Toll-like receptor 4 expression suggests a potential pathway through which ozone generates the inflammatory response in allergic asthmatic subjects but not in atopic subjects without asthma.
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