Genetic Mechanisms Of Susceptibility To Ozone-induced Pulmonary Inflammation

臭氧引起的肺部炎症易感性的遗传机制

• 批准号: 9143469
• 负责人: STEVEN R KLEEBERGER
• 金额: $ 27.7万
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9143469
• 关键词: Acute; Adolescence; Adult; Age; Air Pollutants; Air Pollution; Alabama; Anatomy; Animals; Antioxidants; Attenuated; Biochemical; Breathing; Bronchoalveolar Lavage; Bronchoalveolar Lavage Fluid; C3H/HeJ Mouse; CX3CL1 gene; Candidate Disease Gene; Cell Death; Cell Differentiation process; Cell Surface Receptors; Cells; Child; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 17; Collaborations; Coupled; Cytokine Gene; Development; Edema; Environmental Pollutants; Environmental air flow; Enzyme-Linked Immunosorbent Assay; Epithelial; Etanercept; Event; Exposure to; FGF1 gene; Future; Gene Expression; Gene Expression Profiling; Generations; Genes; Genetic; Genetic Polymorphism; Genetic Predisposition to Disease; Genotype; Goals; Growth and Development function; Homologous Gene; Hour; Human; Hyperplasia; IL17 gene; IL8 gene; Immune; Immune response; Immune system; Inbred Mouse; Inbreeding; Individual; Infant; Infiltration; Inflammation; Inflammatory; Inflammatory Response; Injury; Investigation; Knockout Mice; Lead; Lipids; Lung; Lung Inflammation; MAP Kinase Gene; MHC Class II Genes; Macaca mulatta; Mannose Binding Lectin; Mannose-Binding Lectins; Mediating; Messenger RNA; Microarray Analysis; Minor; Modeling; Molecular Profiling; Monkeys; Mucin-2 Staining Method; Mus; Natural Immunity; Oligonucleotides; Oxidative Stress; Ozone; Pattern; Permeability; Phase; Population; Predisposition; Primates; Proteins; Pulmonary Inflammation; Quantitative Trait Loci; RNA; Receptor Cell; Regimen; Regulation; Relative (related person); Research; Research Personnel; Response Elements; Reverse Transcriptase Polymerase Chain Reaction; Risk; Role; Sampling; Site; Susceptibility Gene; System; TNF gene; Terminal Bronchiole; Testing; Tissues; Toxic effect; Transcript; Universities; Visual; Western Blotting; Wild Type Mouse; air filter; airway inflammation; base; bronchial mucus; clinical investigation; complement pathway; data mining; design; differential expression; gene induction; genome wide association study; human CXCL5 protein; human subject; immune function; in vivo; inhibitor/antagonist; injured airway; injury and repair; insight; lung development; lung injury; lung tumorigenesis; methylation pattern; mouse model; mucus hypersecretion; neutrophil; nonhuman primate; notch protein; novel; oxidation; ozone exposure; positional cloning; pulmonary function; response; transcription factor; transcriptomics

We recently performed studies to reduce the size of Inf2 to determine whether additional candidate genes emerged, and identified Tnf, Lta, Hsp70, and MHC class II genes as important determinants of susceptibility to ozone (O3)-induced airway inflammation. Ongoing studies are investigating the roles of Notch3 and Notch4, also located in Inf2, in O3-induced inflammation. Notch receptors are cell surface receptors important in development and immune cell differentiation. To test the roles of these genes in the model, wild type (B6129SF1, WT), Notch3 (Notch3-/-) and Notch4 (Notch4-/-) knockout mice were exposed to 0.3 ppm O3 or filtered air continuously for 6-48 hr. Immediately after exposure, airway inflammation and injury was assessed using protein concentration (a marker of lung permeability) and inflammatory cells in bronchoalveolar lavage fluid (BALF). O3 significantly increased BALF protein in all genotypes, but greater concentrations were found in Notch3-/- compared to WT (24, 48 hr), and concentrations were greater in Notch4-/- mice compared to Notch3-/- (24, 48 hr). Greater mean numbers of BALF neutrophils were found in Notch3-/- and Notch4-/- mice compared to WT (24, 48 hr). Expression of whole lung Tnf was significantly increased after O3 (24 hr) in all genotypes, and was greater in Notch3-/- and Notch4-/- compared to WT. Pre-treatment with the TNF inhibitor etanercept significantly attenuated the enhanced O3-induced BALF neutrophils in Notch3-/- and Notch4-/- relative to WT. O3-induced transcript expression of other Inf2 genes was not different between genotypes. We then used mRNA transcriptomics analyses to further investigate the role of Notch3/4. Statistical and visual data mining approaches identified differentially expressed genes basally e.g. Gbp1, Cntn1 and after O3 e.g. Ccl7, Il33 between WT and KO mice. Results are consistent with the hypothesis that Notch3 and Notch4 are susceptibility genes for O3-induced airway inflammation. Furthermore, results suggest an important interaction between Notch3, Notch4, and Tnf. These novel findings suggest Notch receptors protect against the innate immune inflammatory response to O3. In another study we have tested the hypothesis that the gene mannose binding lectin (MBL), which has a central role in the activation of the complement pathway of innate immunity, is necessary to elicit some of the pro-inflammatory events caused by ozone-mediated activation of the innate immune system. Our in vivo studies have shown, compared to wild type mice (Mbl+/+), there was significantly less neutrophilic infiltration in the lungs of mice with targeted deletion of Mbl (Mbl-/-) exposed to O3 (0.3 ppm) for 72 hours. We also found reduced levels of the neutrophil attractants MIP-2 and LIX at 48 hours post-exposure in Mbl-/- mice compared to Mbl+/+ mice. Microarray analyses have identified basal and post-exposure profiles and expression response profiles that differ between Mbl+/+ and Mbl-/- mice, providing insight to the mechanisms through which MBL modulates the pulmonary response to O3 inhalation. These novel studies are the first to identify a role for MBL in response of the lung to oxidative stress, and should lead to a better understanding of mechanisms of susceptibility. 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. We are currently investigating methylation patterns in these animals to provide insight to the regulation of the differentially expressed gene transcripts. The transcription factor Nrf2 protects airways from oxidative stress through antioxidant response element-bearing defense gene induction. We designed a study to determine the role of Nrf2 in airway toxicity caused by inhaled O3 in mice. For this purpose, Nrf2-deficient (Nrf2(-/-)) and wild-type (Nrf2(+/+)) mice received acute and subacute exposures to O3. Lung injury was determined by bronchoalveolar lavage and histopathologic analyses. Oxidation markers and mucus hypersecretion were determined by ELISA, and Nrf2 and its downstream effectors were determined by RT-PCR and/or Western blotting. Acute and sub-acute O3 exposures heightened pulmonary inflammation, edema, and cell death more severely in Nrf2(-/-) mice than in Nrf2(+/+) mice. O3 caused bronchiolar and terminal bronchiolar proliferation in both genotypes of mice, while the intensity of compensatory epithelial proliferation, bronchial mucous cell hyperplasia, and mucus hypersecretion was greater in Nrf2(-/-) mice than in Nrf2(+/+) mice. Relative to Nrf2(+/+), O3 augmented lung protein and lipid oxidation more highly in Nrf2(-/-) mice. Results suggest that Nrf2 deficiency exacerbates oxidative stress and airway injury caused by the environmental pollutant O3.

我们最近进行了研究以减少INF2的大小，以确定是否出现了其他候选基因，并确定了TNF，LTA，HSP70和MHC II类基因是对臭氧易感性（O3）诱导的气道炎症的重要决定因素。正在进行的研究正在研究Notch3和Notch4的作用，该Notch3和Notch4也位于INF2，在O3诱导的炎症中。 Notch受体是在发育和免疫细胞分化中重要的细胞表面受体。为了测试这些基因在模型中的作用，将野生型（B6129SF1，WT），Notch3（Notch3 - / - ）和Notch4（Notch4 - / - ）敲除小鼠暴露于0.3 ppm O3或连续过滤空气6-48小时。暴露后，立即使用蛋白质浓度（肺部通透性的标记）和支气管肺泡灌洗液（BALF）评估气道炎症和损伤。在所有基因型中，O3显着增加了BALF蛋白，但是与WT相比（24、48小时），在Notch3 - / - 中发现了更高的浓度，而Notch4 - / - 小鼠的浓度与Notch3 - / - （24、48小时）相比更高。与WT相比，在Notch3 - / - 和Notch4 - / - 小鼠中发现了较大的BALF中性粒细胞数量（24，48小时）。在所有基因型中O3（24小时）后，整个肺TNF的表达显着增加，并且与WT相比，Notch3 - / - 和Notch4 - / - 更大。用TNF抑制剂依然切齿的预处理显着减弱了Notch3 - / - 和Notch4 - / - 相对于WT增强的O3诱导的BALF中性粒细胞。在基因型之间，O3诱导的其他INF2基因的转录本表达没有差异。然后，我们使用mRNA转录组学分析来进一步研究Notch3/4的作用。统计和视觉数据挖掘方法鉴定了差异表达的基因，例如GBP1，CNTN1和O3之后，例如WT和KO小鼠之间的CCL7，IL33。结果与Notch3和Notch4是O3诱导的气道炎症的易感基因的假设一致。此外，结果表明Notch3，Notch4和TNF之间存在重要的相互作用。这些新颖的发现表明Notch受体可以预防对O3的先天免疫炎症反应。 在另一项研究中，我们检验了以下假设：甘露糖结合凝集素（MBL）在激活先天免疫的补体途径中具有核心作用，这对于引起由臭氧介导的先天免疫系统激活引起的一些促炎性事件是必不可少的。我们的体内研究表明，与野生型小鼠（MBL+/+）相比，暴露于O3（0.3 ppm）的MBL（MBL - / - ）的小鼠的肺中嗜中性粒细胞浸润明显较小。我们还发现，与MBL+/+小鼠相比，MBL - / - 小鼠暴露后48小时，中性粒细胞吸引剂MIP-2和Lix降低了水平。 微阵列分析已经确定了MBL+/+和MBL - / - 小鼠之间不同的基础和暴露后谱和表达响应曲线，从而有见识MBL调节对O3吸入的肺反应的机制。这些新的研究是第一个确定MBL在肺对氧化应激的反应中的作用的研究，并应更好地理解易感性的机制。 由于较高的通风率和户外活动水平，儿童可能比成年人更容易受到空气污染的风险，并且持续肺部发展成为青春期。 为了研究O3暴露对发育中的肺的影响，我们与爱德华·邮政局（Univ Alabama，伯明翰大学）合作研究了O3对婴儿鼠尾草猕猴对基因表达的影响，这些猴子猴子的基因表达受到了模仿乌班状况和现场状况和暴露于基因的持续时间和暴露于基因表达分析的方案。 在过滤的空气（FA）中升高灵长类动物，并夜间暴露于1个周期进行的0.5 ppm O3（9 d FA，然后进行8小时/d O3，持续5 d），11个周期或FA。 暴露于180 d年龄。 接触后立即将肺部进行微切除以获得中央轴向气道（8-10代）和没有实质的末端支气管，并储存在Rnalater中。汇总RNA，为每个实验组提供一个样品。 最初通过K-均值聚类分析了基因表达（Agilent Rhesus Monkey Oligo微阵列）。分析了信息模式（Ingenity），以鉴定差异表达基因之间的相互作用。 确定了许多信息模式。 1）1周期后轴向和末端支气管组织上调的基因：炎症和免疫反应（例如IL8，TNF）。 2）11个周期后轴向组织中的基因上调：细胞炎症和血液系统系统（IL1B，IL17，MAPK）。 3）在轴向和末端支气管中差异表达的基因不论O3暴露：发育（ACAN，MUC2，CX3CL1）和免疫功能（IL17RD，FGF1，DAP1）。在婴儿灵长类动物中，损伤和修复对生长和发育的叠加导致基因表达的解剖和暴露特异性改变，这可能与O3诱导的结构，炎症和生化效应偶联。我们目前正在研究这些动物中的甲基化模式，以洞悉调节差异表达基因转录本。 转录因子NRF2通过抗氧化反应元件粘附的防御基因诱导保护气道免受氧化应激。我们设计了一项研究，以确定NRF2在小鼠中吸入O3引起的气道毒性中的作用。为此，NRF2缺陷型（NRF2（ - / - ））和野生型（NRF2（+/+））小鼠接受了O3的急性和亚急性暴露。通过支气管肺泡灌洗和组织病理学分析确定肺损伤。通过ELISA测定氧化标记和粘液过度分泌，NRF2及其下游效应子通过RT-PCR和/或Western blotting确定。与NRF2（+/+）小鼠相比，急性和亚急性O3暴露于NRF2（ - / - ）小鼠中更严重的肺部炎症，水肿和细胞死亡。 O3在两种基因型的小鼠基因型中引起细支气管和末端支气管增生，而NRF2（ - - ）小鼠在NRF2（ - - ）小鼠中的补偿性上皮增殖，支气管粘膜增生和粘液分泌的强度大于NRF2（ - - ）小鼠。相对于NRF2（+/+），O3在NRF2（ - / - ）小鼠中更高的O3增强肺蛋白和脂质氧化。结果表明，NRF2缺乏加剧了由环境污染物O3引起的氧化应激和气道损伤。