Low TET1 Expression Levels in COPD Are Associated with Airway and Blood Neutrophilia

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Linderholm , Maya Juarez , Michael Schivo , Brooks Kuhn , Richart W. Harper , Amir A. Zeki , Angela Haczku doi: https://doi.org/10.1101/2025.04.15.25325889 Hong Ji 1 UC Davis Lung Center, University of California , Davis, CA 2 Department of Anatomy, Physiology and Cell biology; School of Veterinary Medicine , Davis, CA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Hong Ji For correspondence: haczku{at}ucdavis.edu hgji{at}ucdavis.edu Xue Zhang 3 Division of Human Genetics, Cincinnati Children’s Hospital Medical Center , Cincinnati, OH Find this author on Google Scholar Find this author on PubMed Search for this author on this site Angela L. Linderholm 1 UC Davis Lung Center, University of California , Davis, CA 4 Division of Pulmonary, Critical Care, and Sleep Medicine; Department of Internal Medicine, School of Medicine , Davis, CA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Maya Juarez 1 UC Davis Lung Center, University of California , Davis, CA 4 Division of Pulmonary, Critical Care, and Sleep Medicine; Department of Internal Medicine, School of Medicine , Davis, CA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Michael Schivo 1 UC Davis Lung Center, University of California , Davis, CA 4 Division of Pulmonary, Critical Care, and Sleep Medicine; Department of Internal Medicine, School of Medicine , Davis, CA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Brooks Kuhn 1 UC Davis Lung Center, University of California , Davis, CA 4 Division of Pulmonary, Critical Care, and Sleep Medicine; Department of Internal Medicine, School of Medicine , Davis, CA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Richart W. Harper 1 UC Davis Lung Center, University of California , Davis, CA 4 Division of Pulmonary, Critical Care, and Sleep Medicine; Department of Internal Medicine, School of Medicine , Davis, CA 5 Veterans Affairs Medical Center , Mather, CA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Amir A. Zeki 1 UC Davis Lung Center, University of California , Davis, CA 4 Division of Pulmonary, Critical Care, and Sleep Medicine; Department of Internal Medicine, School of Medicine , Davis, CA 5 Veterans Affairs Medical Center , Mather, CA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Angela Haczku 1 UC Davis Lung Center, University of California , Davis, CA 4 Division of Pulmonary, Critical Care, and Sleep Medicine; Department of Internal Medicine, School of Medicine , Davis, CA Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: haczku{at}ucdavis.edu hgji{at}ucdavis.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Epigenetic dysregulation, particularly DNA methylation variations, is implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). Ten-eleven translocation (TET) proteins (TET1, TET2, and TET3) regulate DNA methylation and gene transcription. Impaired TET1 expression was previously associated with airway inflammation and asthma. Here we investigated TET gene associations with COPD severity. We found that reduced TET1 expression in peripheral blood mononuclear cells was associated with higher sputum and blood neutrophil counts, decreased lung function and increased disease severity in patients. These findings support a potential protective role and warrant further mechanistic investigations into the actions of TET1 in COPD. Epigenetic dysregulation is an emerging mechanism implicated in chronic obstructive pulmonary disease (COPD). Variations in DNA methylation are found in genes and pathways associated with COPD etiology and severity 1 , 2 . While the cause of these variations remains unclear, evidence supports the involvement of Ten-eleven translocation methylcytosine dioxygenase (TET) proteins (TET1, TET2, and TET3) known to catalyze the hydroxylation of 5-methylcytosine and regulate DNA methylation. TET proteins are transcriptional regulators of inflammatory, hematopoietic, and oxidative stress related mechanisms, often with differential function 3 . Our lab previously showed that TET1 inhibits allergic airway inflammation 4 , while studies in patients and mouse models of COPD implied TET2 as protective 5 , 6 . Interestingly, TET1 expression was reduced in COPD both in the lungs of patients and mouse models 2 , 7 However, the role of TET1 in COPD remains unclear. Here we examined the associations between the expression of TET family genes with airway neutrophilia, inflammatory and oxidative stress pathways and COPD severity. We used bulk RNA-seq assessment of peripheral blood mononuclear cell (PBMC) samples from a cohort of healthy subjects (N=15) and COPD patients (N=17). Lung function measurements, differential cell counts in blood and induced sputum were also performed on these subjects. Detailed recruitment, sample processing and data acquisition are described in the Supplemental Material and Methods section. COPD patients showed a significantly decreased lung function and elevated neutrophils in the sputum and blood (Supplementary Table 1). Sputum neutrophils (%) negatively associated with lung function in all participants (FEV1, FEV1% predicted, and FEV1/FVC) and in COPD patients (FEV1) ( Table 1 ). Neutrophils positively associated with higher COPD Assessment Test (CAT) scores in all participants (p=0.001) and in COPD patients (p=0.172) ( Figure 1A and Table 1 ). Higher CAT scores indicate greater symptom burden. Blood neutrophil percentage and absolute counts were also negatively associated with lung function and positively associated with CAT in all participants ( Table 1 ). View this table: View inline View popup Download powerpoint Table 1: Correlations between sputum and blood neutrophil parameters with lung function and CAT. Download figure Open in new tab Figure 1. Relationships between Neutrophil counts, blood TET normalized expression values and COPD and implicated mechanisms. A . Sputum neutrophil% (after squamous cell removal) positively correlates with CAT scores. B . Association between TET1 expression with sputum neutrophils% and blood neutrophils (NEUTROPHILx10 3 /uL). C . TET1 expression in PBMC from healthy subjects and COPD patients. D . Logistic regression plots showing the prediction of COPD by TET1 expression level and blood neutrophil concentration. Logistic regression models were applied. E . Correlation between TET1 and IRF1, NFKBIZ expression levels in PBMC. F . Expression levels of NFKBIZ, CYP1B1 and AHR in PBMC from healthy subjects and COPD patients. G . Associations of IRF1, NFKBIZ, and CYP1B1 expression levels in PBMC with sputum neutrophil%. H . Diagram of proposed model. In healthy subjects, TET1 in PBMCs limits the expression of cytokines and transcription factors, likely by controlling chromatin and histone modification instead of DNA methylation. In COPD patients, TET1 expression is low and TET2 and/or TET3 would replace TET1 and bind to the promoter of these genes, remove DNA methylation and promote their expression. For expression of genes, normalized expression values from RNA-seq are plotted. We discovered that TET1 mRNA expression was negatively associated with sputum neutrophil % and blood neutrophil concentration ( Figure 1B and Table 2 , p=0.080 and p=0.026 after adjusting for age). The negative relationship remained when stratified by COPD status (e.g., p=0.039 for blood NEUTROPHIL x10 3 /uL) in healthy subjects; p=0.178 for sputum neutrophil % in COPD). Consistent with previous observations, there was lower TET1 expression in the peripheral blood of COPD patients when compared with healthy subjects ( Figure 1C ). Importantly, age showed no significant contribution (p=0.955) and was excluded from the statistical model. Additionally, TET1 significantly negatively correlated with TET3 (p=0.002) while TET2 and TET3 expression showed a positive correlation with one another (p=0.026), suggesting differential regulation and function of these genes. Although we did not find statistically significant associations between blood TET2 or TET3 expression and COPD, we observed significant positive associations of blood TET2 and TET3 with blood neutrophil counts. No significant associations between TET1, TET2 and TET3 expression and lung function were found. View this table: View inline View popup Download powerpoint Table 2. Associations of blood TET mRNA levels with clinical outcomes. Logistical regression analyses revealed that higher TET1 expression was associated with a reduced odds of having COPD (odds ratio of 0.520, [95% CI, (0.250, 0.936)] ( Figure 1D , p=0.036), suggesting that higher TET1 expression may be protective. Consistent with the critical role of neutrophils in COPD pathogenesis, we found that higher blood neutrophil counts and sputum neutrophil percentages were associated with an increase in the odds of having COPD [odds ratio=4.78, 95% CI (1.72, 26.62), p=0.002; and odds ratio=1.053, 95% CI (1.018 to 1.102), p=0.004] ( Figure 1D ). Since sputum neutrophil percent and blood neutrophil counts were also associated with TET1 expression ( Table 2 ), we used logistic models with and without different neutrophil parameters to investigate whether the impact of TET1 on COPD may be mediated by neutrophils. We found that the effects (regression coefficients) of TET1 on COPD decreased when these factors were added to the models as the mediator, suggesting potential mediation by these factors, though none of them reached the statistical significance in the causal inference test (p=0.08-0.11, Supplementary Table 2). These data suggest that TET1 may limit neutrophil counts. While our study sample size is small, as a discovery cohort this information is useful for hypothesis generation and to guide future investigations. Our prior studies in human airway epithelial cells support a new role of TET1 in limiting cytokine production through histone modification and chromatin accessibility 8 . The TET proteins may interact with other epigenetic regulators and regulate the expression of genes important for neutrophil function and inflammatory (inflammasome and NF-kB) pathways. TET proteins, additionally, have a complex relationship with the Aryl Hydrocarbon Receptor (AhR), a major regulator of oxidative stress. In fact, AhR activation may induce TET2 expression through promoter binding and interacting with TET2 as transcriptional co-regulators in immune cell lineage specificity during inflammation 9 . In support of potential differential functions between TET1 and TET2, we observed significant negative correlations between TET1 mRNA with IRF1 and NFKBIZ ( Figure 1E ), but positive correlations between TET2 with IRF1 and NFKBIZ , and positive correlations between TET3 with IRF1 and NFKBIZ (Supplementary Figure 1). TET2, TET3 and ARNT also significantly positively correlated with IL1B while TET1 showed a negative trend (p=0.281) (Supplementary Figure 1). Importantly, IRF1, NFKBIZ, CYP1B1 and AHR were upregulated in COPD patients ( Figure 1F ). Sputum neutrophils (%) were positively associated with IRF1, NFKBIZ and CYP1B1 (a direct target of the AhR receptor) ( Figure 1G ). Our study provides intriguing new findings supporting a protective role for TET1 in COPD potentially through limiting neutrophilia. Decreased peripheral blood cell TET1 expression was associated with higher CAT scores and increased sputum neutrophil percentage. Neutrophils and their products are key contributors to airway inflammation in COPD, and cause many of the disease-associated pathological features, including emphysema and mucus hypersecretion 10 . Reduced TET1 expression was also associated with an increase of pro-neutrophilic inflammatory genes (e.g., IL1B, IRF1, NFKBIZ ) ( Figure 1H ). TET proteins may play differential and possibly opposing roles in regulating the same set of genes in COPD through distinct mechanisms ( Figure 1H ). Additional mechanistic studies are needed to substantiate these novel observations and decipher how TET1, TET2 and TET3 function in COPD and other chronic airways diseases. Declarations Ethics approval and consent to participate This study was reviewed and approved by the University of California, Davis Institutional Review Board. Data Availability All data produced in the present work are available upon reasonable request to the authors. Competing interests The authors have declared no competing interests. Funding This work was supported by American Lung Association Innovation Award (IA-1264831) and NIH/NIAID R01AI141569 to H.J., Chester Robbins Pulmonary Research Endowment and TRDRP 27IR-0053 to A.H. Authors Contributions H.J. and A.H. conceived the study. H.J. performed data analysis with the assistance of X.Z. A.L.L., M.J., M.S., B.K., R.W.H., and A.A.Z. planned and performed clinical studies. H.J., A.A.Z., and A.H. wrote the manuscript. Footnotes Funding information has been added in the manuscript References 1. ↵ Song J , Heijink IH , Kistemaker LEM , et al. Aberrant DNA methylation and expression of SPDEF and FOXA2 in airway epithelium of patients with COPD . Clin Epigenetics . 2017 ; 9 : 42 . doi: 10.1186/s13148-017-0341-7 OpenUrl CrossRef PubMed 2. ↵ Yeung-Luk BH , Wally A , Swaby C , et al. Epigenetic Reprogramming Drives Epithelial Disruption in Chronic Obstructive Pulmonary Disease . Am J Respir Cell Mol Biol . Mar 2024 ; 70 ( 3 ): 165 – 177 . doi: 10.1165/rcmb.2023-0147OC OpenUrl CrossRef PubMed 3. ↵ Rasmussen KD , Helin K. Role of TET enzymes in DNA methylation, development, and cancer . Genes Dev . Apr 1 2016 ; 30 ( 7 ): 733 – 50 . doi: 10.1101/gad.276568.115 OpenUrl Abstract / FREE Full Text 4. ↵ Burleson JD , Siniard D , Yadagiri VK , et al. TET1 contributes to allergic airway inflammation and regulates interferon and aryl hydrocarbon receptor signaling pathways in bronchial epithelial cells . Sci Rep . May 14 2019 ; 9 ( 1 ): 7361 . doi: 10.1038/s41598-019-43767-6 OpenUrl CrossRef PubMed 5. ↵ Zeng Z , Li T , Liu X , et al. DNA dioxygenases TET2 deficiency promotes cigarette smoke induced chronic obstructive pulmonary disease by inducing ferroptosis of lung epithelial cell . Redox Biol . Nov 2023 ; 67 : 102916 . doi: 10.1016/j.redox.2023.102916 OpenUrl CrossRef PubMed 6. ↵ Miller PG , Qiao D , Rojas-Quintero J , et al. Association of clonal hematopoiesis with chronic obstructive pulmonary disease . Blood . Jan 20 2022 ; 139 ( 3 ): 357 – 368 . doi: 10.1182/blood.2021013531 OpenUrl CrossRef PubMed 7. ↵ Ghosh AJ , Hobbs BD , Yun JH , et al. Lung tissue shows divergent gene expression between chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis . Respir Res . Apr 21 2022 ; 23 ( 1 ): 97 . doi: 10.1186/s12931-022-02013-w OpenUrl CrossRef PubMed 8. ↵ Brown AP , Parameswaran S , Cai L , et al. TET1 regulates responses to house dust mite by altering chromatin accessibility, DNA methylation, and gene expression in airway epithelial cells . Res Sq . Dec 13 2023 ; doi: 10.21203/rs.3.rs-3726852/v1 OpenUrl CrossRef 9. ↵ Cheng HH , Lin HK , Sheu ML , Lee CY , Tsai YC , Lai DW . AHR/TET2/NT5E axis downregulation is associated with the risk of systemic lupus erythematosus and its progression . Immunology . Apr 2023 ; 168 ( 4 ): 654 – 670 . doi: 10.1111/imm.13600 OpenUrl CrossRef 10. ↵ Jasper AE , McIver WJ , Sapey E , Walton GM . Understanding the role of neutrophils in chronic inflammatory airway disease . F1000Res . 2019 ; 8 doi: 10.12688/f1000research.18411.1 OpenUrl CrossRef View the discussion thread. Back to top Previous Next Posted May 05, 2025. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about medRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. 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Linderholm , Maya Juarez , Michael Schivo , Brooks Kuhn , Richart W. Harper , Amir A. 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