Genetic and histologic evidence for autophagy in asthma pathogenesis
2011; Elsevier BV; Volume: 129; Issue: 2 Linguagem: Inglês
10.1016/j.jaci.2011.09.035
ISSN1097-6825
AutoresAudrey H. Poon, Fazila Chouiali, Sze Man Tse, Augusto A. Litonjua, Sabah N. A. Hussain, Carolyn J. Baglole, David H. Eidelman, Ronald Olivenstein, James G. Martin, Scott T. Weiss, Qutayba Hamid, Catherine Laprise,
Tópico(s)Chronic Obstructive Pulmonary Disease (COPD) Research
ResumoAsthma affects all age groups and presents itself as a spectrum of severity and symptoms. Reactive oxygen species (ROS) play a pivotal role in asthma pathogenesis. Exhaled levels of mediators associated with ROS positively correlate with asthma severity.1Horvath I. Donnelly L.E. Kiss A. Kharitonov S.A. Lim S. Chung K.F. et al.Combined use of exhaled hydrogen peroxide and nitric oxide in monitoring asthma.Am J Respir Crit Care Med. 1998; 158: 1042-1046Crossref PubMed Scopus (232) Google Scholar Autophagy, the process of cellular waste disposal through lysosome-dependent pathways, is induced by ROS to remove oxidized proteins or organelles to minimize tissue damage.2Szumiel I. Autophagy, reactive oxygen species and the fate of mammalian cells.Free Radic Res. 2011; 45: 253-265Crossref PubMed Scopus (49) Google Scholar Although autophagy is augmented in the lungs of patients with chronic obstructive pulmonary disease compared with healthy control subjects,3Chen Z.H. Kim H.P. Sciurba F.C. Lee S.J. Feghali-Bostwick C. Stolz D.B. et al.Egr-1 regulates autophagy in cigarette smoke-induced chronic obstructive pulmonary disease.PLoS One. 2008; 3: e3316Crossref PubMed Scopus (365) Google Scholar evidence for autophagy in asthmatic patients, particularly those with moderate-to-severe asthma, has not been reported. We hypothesize that autophagy is associated with asthma pathogenesis and sought to detect its presence using both genetic and histologic approaches.We conducted a genetic association study to investigate whether single nucleotide polymorphisms (SNPs) in genes of the autophagy pathway are associated with asthma. We selected 5 genes of the autophagy pathway (unc-51-like kinase 1 [ULK1], sequestosome 1 [SQSTM1], microtubule-associated protein 1 light chain 3β [MAP1LC3B], beclin 1 [BECN1], and autophagy-related 5 homolog [ATG5]).2Szumiel I. Autophagy, reactive oxygen species and the fate of mammalian cells.Free Radic Res. 2011; 45: 253-265Crossref PubMed Scopus (49) Google Scholar We tested for genetic associations in an asthma family-based study from northeastern Quebec, Canada (the Saguenay-Lac-Saint-Jean [SLSJ] asthma study), by using the family-based association test statistic and UNPHASED software for an odds ratio estimate.4Dudbridge F. Likelihood-based association analysis for nuclear families and unrelated subjects with missing genotype data.Hum Hered. 2008; 66: 87-98Crossref PubMed Scopus (542) Google Scholar, 5Lake S.L. Blacker D. Laird N.M. Family-based tests of association in the presence of linkage.Am J Hum Genet. 2000; 67: 1515-1525Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar Patient recruitment has previously been described.6Moffatt M.F. Gut I.G. Demenais F. Strachan D.P. Bouzigon E. Heath S. et al.A large-scale, consortium-based genomewide association study of asthma.N Engl J Med. 2010; 363: 1211-1221Crossref PubMed Scopus (1450) Google Scholar These SNPs have been genotyped previously in a genome-wide association study.6Moffatt M.F. Gut I.G. Demenais F. Strachan D.P. Bouzigon E. Heath S. et al.A large-scale, consortium-based genomewide association study of asthma.N Engl J Med. 2010; 363: 1211-1221Crossref PubMed Scopus (1450) Google Scholar To reduce the likelihood of false-positive findings, we reset the statistical significance threshold from a P value of .05 to a P value of .001, according to the Bonferroni method. To confirm our positive findings, we tested the association in a second family-based population, the non-Hispanic white participants of the Childhood Asthma Management Program (CAMP), and patient recruitment has previously been described.7The Childhood Asthma Management Program (CAMP): design, rationale, and methods. Childhood Asthma Management Program Research Group.Control Clin Trials. 1999; 20: 91-120Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar The SLSJ local ethics committee and the Institutional Review Board for CAMP approved the study, and all subjects provided informed consent.In the SLSJ asthma study a total of 1338 subjects (483 nuclear families) with known asthma status were included in the analysis, and 336 subjects were either probands or affected siblings. Of this group, the male/female ratio was 0.83. The mean age was 16.45 years (SD, ±9.43 years), and 77.1% were atopic. The mean FEV1 percent predicted value was 94.1% (SD, 20.1%). A total of 39 SNPs were tested, and after Bonferroni correction, SNP rs12212740 G>A of ATG5 remained statistically significant (Table I). Allele G, with an allele frequency of 0.88, is overtransmitted to asthmatic offspring (P = .0002; odds ratio, 1.35; 95% CI, 1.01-1.89). SNP rs12212740 was not associated with asthma in CAMP; however, it was associated with prebronchodilator FEV1 (adjusted for age, sex, and height; P = .04). In the SLSJ cohort rs12212740 was associated with prebronchodilator FEV1 (percent predicted; P = .007). In both populations allele G was negatively associated with adjusted prebronchodilator FEV1. SNP rs12212740 is located in intron 3 of ATG5, which is located 7 kb downstream and 8 kb upstream of exons 3 and 4, respectively. At present, the functional consequence of SNP rs12212740 is unknown, and it is probable that the association is due to the linkage disequilibrium between SNP rs12212740 and the true causative variant. Nevertheless, the association between a genetic variant of ATG5 and prebronchodilator FEV1 in both study populations suggests that autophagy is associated with reduced lung function in asthmatic patients.Table IAssociation of SNPs and asthma in the SLSJ asthma studyGeneSNPAlleleAllele frequencyNo. of informative familiesP valueBECN1rs10512488A0.82180.1019MAP1LC3Brs7204722T0.82167.3834rs8051218T0.9564.141rs2873702A0.9919−.9351rs2241617T0.87118.8613rs3748400T0.75196.8694SQSTM1rs166624G0.86131−.5718rs10516140C0.8168.9025rs565280G0.59224.9447rs155788T0.9197−.1876rs10277C0.6221.8631rs1065154T0.6222.8886rs248244C0.9560−.7241rs248247G0.62232−.7656rs248248C0.78198.4782rs2303677C0.78180−.2451rs155793C0.9191−.6668rs748197G0.55232.3494ULK1rs11246867G0.9568.0187rs9652059C0.85148.0126rs10902472C0.9564.0387rs7487166G0.87132.036ATG5rs9398071T0.77176−.0694rs6920944C0.9177.9309rs10484575C0.9287−.8106rs9386514T0.8164−.1043rs9486306T0.87117−.2731rs1769972T0.9561.739rs3804332T0.9665−.4294rs3851210C0.9482.2755rs3804333C0.78174−.1332rs633724C0.66222.0145rs9486314C0.82143−.1427rs12529626A0.9107.0627rs10485352A0.9665−.4297rs12212740∗After Bonferroni correction, SNP rs12212740 of ATG5 remained statistically significant.G0.88130.0002rs1766208C0.88132−.5169rs3761796A0.9915.3656rs2355380T0.7220.2373Thirty-nine SNPs were tested for association with asthma in the SLSJ asthma study. The gene symbols, rs numbers for each SNP, major alleles of each SNP, numbers of informative families that contributed to the test statistic, and unadjusted P values are listed. Positive P values represent overtransmission of the major allele to affected offspring, and negative P values represent undertransmission.∗ After Bonferroni correction, SNP rs12212740 of ATG5 remained statistically significant. Open table in a new tab Bronchial biopsy specimens stored at the Tissue Bank of the Respiratory Health Network of the Fonds de la Recherché en Santé du Québec (McGill University Health Centre site) were obtained to determine whether autophagy is present in the airways of asthmatic patients. Patient recruitment and bronchoscopy have previously been described.8Shannon J. Ernst P. Yamauchi Y. Olivenstein R. Lemiere C. Foley S. et al.Differences in airway cytokine profile in severe asthma compared to moderate asthma.Chest. 2008; 133: 420-426Crossref PubMed Scopus (180) Google Scholar Bronchial biopsy tissue from a patient with moderately severe asthma and a healthy control subject were viewed by means of electron microscopy (EM) for double-membrane autophagosomes.Here we demonstrated evidence of autophagy in asthma pathogenesis using EM in a tissue sample from a patient with moderately severe asthma. By using EM, double-membrane autophagosomes were detected in fibroblasts and epithelial cells. A representative fibroblast from a bronchial biopsy tissue of a patient with moderate asthma is depicted in Fig 1, A to C, and epithelial cells are depicted in Fig 2, A to C. Corresponding fibroblast and epithelial cells from a healthy control subject are depicted in Fig 1, D to F, and Fig 2, D to F, respectively, where fewer or no autophagosome were detected.Fig 2Autophagosomes were detected in bronchial epithelial cells from a patient with moderate asthma. Tissue was viewed at magnifications of ×4030 (A), ×9760 (B), and ×37,000 (C), respectively. Corresponding epithelial cells of a healthy subject were viewed at magnifications of ×6,390 (D), ×16,100 (E), and ×37,000 (F), respectively. Arrows indicate double-membrane autophagosomes.View Large Image Figure ViewerDownload Hi-res image Download (PPT)This is the first report to present genetic and histologic evidence of autophagy in asthma pathogenesis. ATG5 is involved in the elongation step of the autophagosome formation. ATG5 forms a complex with ATG12 and ATG16L1, and the complexes are found on the outer membrane of the forming autophagosome.2Szumiel I. Autophagy, reactive oxygen species and the fate of mammalian cells.Free Radic Res. 2011; 45: 253-265Crossref PubMed Scopus (49) Google Scholar We speculate that the positive association of allele G with asthma and the negative association with prebronchodilator FEV1 in asthmatic patients might be due to the inverse relationship between prebronchodilator FEV1 and asthma severity.9Lange P. Parner J. Vestbo J. Schnohr P. Jensen G.A. 15-year follow-up study of ventilatory function in adults with asthma.N Engl J Med. 1998; 339: 1194-1200Crossref PubMed Scopus (1077) Google Scholar If allele G is a risk factor for low prebronchodilator FEV1, given that prebronchodilator FEV1 tends to be less in those with more severe forms of asthma, the allele would also increase the risk of moderate-to-severe asthma. The genetic association of ATG5 with prebronchodilator FEV1 in asthmatic patients and the detection of autophagosomes in fibroblasts and epithelial cells in tissues from a patient with moderately severe asthma suggest an association between autophagy and reduced lung function in asthmatic patients. At present, the mechanistic pathway of autophagy in asthma is unclear, but it opens up a new avenue to explore the mechanism of the chronic nature of asthma pathogenesis. Asthma affects all age groups and presents itself as a spectrum of severity and symptoms. Reactive oxygen species (ROS) play a pivotal role in asthma pathogenesis. Exhaled levels of mediators associated with ROS positively correlate with asthma severity.1Horvath I. Donnelly L.E. Kiss A. Kharitonov S.A. Lim S. Chung K.F. et al.Combined use of exhaled hydrogen peroxide and nitric oxide in monitoring asthma.Am J Respir Crit Care Med. 1998; 158: 1042-1046Crossref PubMed Scopus (232) Google Scholar Autophagy, the process of cellular waste disposal through lysosome-dependent pathways, is induced by ROS to remove oxidized proteins or organelles to minimize tissue damage.2Szumiel I. Autophagy, reactive oxygen species and the fate of mammalian cells.Free Radic Res. 2011; 45: 253-265Crossref PubMed Scopus (49) Google Scholar Although autophagy is augmented in the lungs of patients with chronic obstructive pulmonary disease compared with healthy control subjects,3Chen Z.H. Kim H.P. Sciurba F.C. Lee S.J. Feghali-Bostwick C. Stolz D.B. et al.Egr-1 regulates autophagy in cigarette smoke-induced chronic obstructive pulmonary disease.PLoS One. 2008; 3: e3316Crossref PubMed Scopus (365) Google Scholar evidence for autophagy in asthmatic patients, particularly those with moderate-to-severe asthma, has not been reported. We hypothesize that autophagy is associated with asthma pathogenesis and sought to detect its presence using both genetic and histologic approaches. We conducted a genetic association study to investigate whether single nucleotide polymorphisms (SNPs) in genes of the autophagy pathway are associated with asthma. We selected 5 genes of the autophagy pathway (unc-51-like kinase 1 [ULK1], sequestosome 1 [SQSTM1], microtubule-associated protein 1 light chain 3β [MAP1LC3B], beclin 1 [BECN1], and autophagy-related 5 homolog [ATG5]).2Szumiel I. Autophagy, reactive oxygen species and the fate of mammalian cells.Free Radic Res. 2011; 45: 253-265Crossref PubMed Scopus (49) Google Scholar We tested for genetic associations in an asthma family-based study from northeastern Quebec, Canada (the Saguenay-Lac-Saint-Jean [SLSJ] asthma study), by using the family-based association test statistic and UNPHASED software for an odds ratio estimate.4Dudbridge F. Likelihood-based association analysis for nuclear families and unrelated subjects with missing genotype data.Hum Hered. 2008; 66: 87-98Crossref PubMed Scopus (542) Google Scholar, 5Lake S.L. Blacker D. Laird N.M. Family-based tests of association in the presence of linkage.Am J Hum Genet. 2000; 67: 1515-1525Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar Patient recruitment has previously been described.6Moffatt M.F. Gut I.G. Demenais F. Strachan D.P. Bouzigon E. Heath S. et al.A large-scale, consortium-based genomewide association study of asthma.N Engl J Med. 2010; 363: 1211-1221Crossref PubMed Scopus (1450) Google Scholar These SNPs have been genotyped previously in a genome-wide association study.6Moffatt M.F. Gut I.G. Demenais F. Strachan D.P. Bouzigon E. Heath S. et al.A large-scale, consortium-based genomewide association study of asthma.N Engl J Med. 2010; 363: 1211-1221Crossref PubMed Scopus (1450) Google Scholar To reduce the likelihood of false-positive findings, we reset the statistical significance threshold from a P value of .05 to a P value of .001, according to the Bonferroni method. To confirm our positive findings, we tested the association in a second family-based population, the non-Hispanic white participants of the Childhood Asthma Management Program (CAMP), and patient recruitment has previously been described.7The Childhood Asthma Management Program (CAMP): design, rationale, and methods. Childhood Asthma Management Program Research Group.Control Clin Trials. 1999; 20: 91-120Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar The SLSJ local ethics committee and the Institutional Review Board for CAMP approved the study, and all subjects provided informed consent. In the SLSJ asthma study a total of 1338 subjects (483 nuclear families) with known asthma status were included in the analysis, and 336 subjects were either probands or affected siblings. Of this group, the male/female ratio was 0.83. The mean age was 16.45 years (SD, ±9.43 years), and 77.1% were atopic. The mean FEV1 percent predicted value was 94.1% (SD, 20.1%). A total of 39 SNPs were tested, and after Bonferroni correction, SNP rs12212740 G>A of ATG5 remained statistically significant (Table I). Allele G, with an allele frequency of 0.88, is overtransmitted to asthmatic offspring (P = .0002; odds ratio, 1.35; 95% CI, 1.01-1.89). SNP rs12212740 was not associated with asthma in CAMP; however, it was associated with prebronchodilator FEV1 (adjusted for age, sex, and height; P = .04). In the SLSJ cohort rs12212740 was associated with prebronchodilator FEV1 (percent predicted; P = .007). In both populations allele G was negatively associated with adjusted prebronchodilator FEV1. SNP rs12212740 is located in intron 3 of ATG5, which is located 7 kb downstream and 8 kb upstream of exons 3 and 4, respectively. At present, the functional consequence of SNP rs12212740 is unknown, and it is probable that the association is due to the linkage disequilibrium between SNP rs12212740 and the true causative variant. Nevertheless, the association between a genetic variant of ATG5 and prebronchodilator FEV1 in both study populations suggests that autophagy is associated with reduced lung function in asthmatic patients. Thirty-nine SNPs were tested for association with asthma in the SLSJ asthma study. The gene symbols, rs numbers for each SNP, major alleles of each SNP, numbers of informative families that contributed to the test statistic, and unadjusted P values are listed. Positive P values represent overtransmission of the major allele to affected offspring, and negative P values represent undertransmission. Bronchial biopsy specimens stored at the Tissue Bank of the Respiratory Health Network of the Fonds de la Recherché en Santé du Québec (McGill University Health Centre site) were obtained to determine whether autophagy is present in the airways of asthmatic patients. Patient recruitment and bronchoscopy have previously been described.8Shannon J. Ernst P. Yamauchi Y. Olivenstein R. Lemiere C. Foley S. et al.Differences in airway cytokine profile in severe asthma compared to moderate asthma.Chest. 2008; 133: 420-426Crossref PubMed Scopus (180) Google Scholar Bronchial biopsy tissue from a patient with moderately severe asthma and a healthy control subject were viewed by means of electron microscopy (EM) for double-membrane autophagosomes. Here we demonstrated evidence of autophagy in asthma pathogenesis using EM in a tissue sample from a patient with moderately severe asthma. By using EM, double-membrane autophagosomes were detected in fibroblasts and epithelial cells. A representative fibroblast from a bronchial biopsy tissue of a patient with moderate asthma is depicted in Fig 1, A to C, and epithelial cells are depicted in Fig 2, A to C. Corresponding fibroblast and epithelial cells from a healthy control subject are depicted in Fig 1, D to F, and Fig 2, D to F, respectively, where fewer or no autophagosome were detected. This is the first report to present genetic and histologic evidence of autophagy in asthma pathogenesis. ATG5 is involved in the elongation step of the autophagosome formation. ATG5 forms a complex with ATG12 and ATG16L1, and the complexes are found on the outer membrane of the forming autophagosome.2Szumiel I. Autophagy, reactive oxygen species and the fate of mammalian cells.Free Radic Res. 2011; 45: 253-265Crossref PubMed Scopus (49) Google Scholar We speculate that the positive association of allele G with asthma and the negative association with prebronchodilator FEV1 in asthmatic patients might be due to the inverse relationship between prebronchodilator FEV1 and asthma severity.9Lange P. Parner J. Vestbo J. Schnohr P. Jensen G.A. 15-year follow-up study of ventilatory function in adults with asthma.N Engl J Med. 1998; 339: 1194-1200Crossref PubMed Scopus (1077) Google Scholar If allele G is a risk factor for low prebronchodilator FEV1, given that prebronchodilator FEV1 tends to be less in those with more severe forms of asthma, the allele would also increase the risk of moderate-to-severe asthma. The genetic association of ATG5 with prebronchodilator FEV1 in asthmatic patients and the detection of autophagosomes in fibroblasts and epithelial cells in tissues from a patient with moderately severe asthma suggest an association between autophagy and reduced lung function in asthmatic patients. At present, the mechanistic pathway of autophagy in asthma is unclear, but it opens up a new avenue to explore the mechanism of the chronic nature of asthma pathogenesis. We thank Glenda Wright, Louise Pelletier, and Anja Geitmann for their assistance in EM.
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