研究背景

难治性哮喘(RA)患者占哮喘病人的3%-10%，占哮喘相关医疗保健利用率的60%以上，尽管坚持以指南为基础的治疗，但其症状控制仍较差。研究发现，与健康对照组(C)或非难治性哮喘(NR)患者相比，RA患者的呼吸道促嗜中性粒细胞增多，这与亚临床感染/炎症(SBI)有关，其潜在机制尚不清楚。

研究发现

1. 刷检和活检样本的比较发现RA刷检中有更多下调的差异表达基因

刷检和活组织检查样本有151个重叠的差异表达基因(DEG)，其中141个在RA中下调,10个上调。下调基因中110个已知富含纤毛或成熟的纤毛细胞，82个被鉴定为早期纤毛、晚期纤毛或成熟纤毛状态，35个在纤毛细胞中具有功能验证的作用。

2. 等级聚类法确定纤毛相关基因表达减少和呼吸道炎症增加的RA亚组

刷检和活检数据的单独聚类分析显示，在刷检或活检样本中，有一组RA(34名受试者)纤毛细胞基因下调，将其标记为纤毛缺陷RA(CDRA)。经年龄、性别、BMI和口服皮质类固醇(OCS)调整后，CDRA与其他RA受试者相比，有更高的SBI几率;更高的血液、活检和支气管肺泡灌洗液(BALF)嗜酸性粒细胞;以及更高的BALF中性粒细胞。

回顾60名RA受试者中58名的胸部CT报告，并根据“粘液”、“支气管扩张”或“树芽”描述的表现进行分类。34名CDRA受试者中有23人至少有1项发现。26名非CDRA受试者中，24名有CT报告，只有8人有描述的模式，这表明纤毛相关基因减少与具有临床意义的气道粘液模式增加有关。

3. 加权基因相关网络分析加强DEG分析

加权基因相关网络分析(WGCNA)的发现类似于差异表达分析，发现与纤毛功能相关的共表达网络在RA和其他RA之间存在显著差异。WGCNA对刷检和活检的数据分析显示，下调的基因模块富含与纤毛组装、组织和运动相关的基因，而大多数上调的模块富含与免疫反应相关的基因。

结 论

尽管该项研究具有局限性和探索性，但研究者们确定了一个独特的、临床上重要的纤毛缺陷难治性哮喘亚群。纤毛相关基因的表达减少与较高的粘液负担和亚临床细菌感染有关，可能是哮喘难以治疗的原因之一。

讨 论

1. 负责纤毛细胞分化、轴丝形成和定向运动的基因在我们称为CDRA的RA亚群中下调。这一亚组更有可能出现SBI、CT影像上较高的呼吸道炎症和较低的FEV1。据研究者所知，他们首次将CDRA描述为难治性中性粒细胞性哮喘的一个亚群。

2. 这项研究有几个局限性。(1)基因表达的群体差异也可能受到哮喘，特别是RA自然病史的影响。虽然在分析中根据年龄进行了调整，但与NR相比，RA的年龄更高，病情恶化的几率更高。(2)所有NR患者都在接受吸入皮质类固醇(ICS)治疗，剂量差异可能会潜在影响结果,其他研究小组已经发现ICS可以改变呼吸道上皮基因的表达。然而，作者注意到CDRA组甚至与另一组RA组相比纤毛相关基因的表达显著减少，尽管这两组具有相似的临床特征、病情恶化、OCS接收和统一的高ICS剂量。(3)对于大量的基因表达数据，作者不能完全肯定地说，CDRA组纤毛相关基因的下调是由于CDRA纤毛细胞表达受抑，还是由于纤毛细胞丢失;然而，这两种情况中的任何一种都可能表明纤毛功能受损，并导致难治性。

3. 未来的研究还应纠正吸入类固醇和β-激动剂对纤毛基因表达和功能的影响。临床上相关的纤毛失调特征也需要在包括T2和非T2哮喘的机制研究中得到证实。更高的SBI、更高的粘液负荷、CT的支气管壁扩张和增厚影像，以及纤毛失调导致的呼吸道清除不良，都可能致使CDRA中哮喘控制更差，需要进一步的系统研究。

4. 鼻腔上皮细胞的基因表达谱与下呼吸道上皮细胞的基因表达谱有很大重叠。鼻腔上皮细胞纤毛相关基因可通过实时定量聚合酶链式反应或免疫染色进行检测。作者预计鼻腔上皮细胞纤毛相关基因的表达会较低。如果得到证实，那么鼻拭子的检查可以帮助指导CDRA类型的检测和通过基因表达谱分析治疗反应。

参考文献

1. Israel E, Reddel HK. Severe and difficult-to-treat asthma in adults. N Engl J Med 2017;377:965-76.

2. Chung KF, Wenzel SE, Brozek JL, Bush A, Castro M, Sterk PJ, et al. International ERS/ATS guidelines on definition, evaluation and treatment of severe asthma. Eur Respir J 2014;43:343-73.

3. Alam R, Good J, Rollins D, V erma M, Chu H, Pham TH, et al. Airway and serum biochemical correlates of refractory neutrophilic asthma. J Allergy Clin Immunol 2017;140:1004-14.e13.

4. Good JT Jr, Kolakowski CA, Groshong SD, Murphy JR, Martin RJ. Refractory asthma: importance of bronchoscopy to identify phenotypes and direct therapy.Chest 2012;141:599-606.

5. Liu W, Liu S, V erma M, Zafar I, Good JT, Rollins D, et al. Mechanism of TH2/ TH17-predominant and neutrophilic TH2/TH17-low subtypes of asthma. J Allergy Clin Immunol 2017;139:1548-58.e4.

6. Modena BD, Bleecker ER, Busse WW, Erzurum SC, Gaston BM, Jarjour NN, et al.Gene expression correlated with severe asthma characteristics reveals heterogeneous mechanisms of severe disease. Am J Respir Crit Care Med 2017;195: 1449-63.

7. Gomez JL, Chen A, Diaz MP , Zirn N, Gupta A, Britto C, et al. A network of sputum microRNAs is associated with neutrophilic airway inflammation in asthma.Am J Respir Crit Care Med 2020;202:51-64.

8. Holgate ST, Holloway J, Wilson S, Bucchieri F, Puddicombe S, Davies DE. Epithelial–mesenchymal communication in the pathogenesis of chronic asthma. Proc Am Thorac Soc 2004;1:93-8.

9. Potaczek DP, Miethe S, Schindler V , Alhamdan F, Garn H. Role of airway epithelial cells in the development of different asthma phenotypes. Cell Signal 2020;69: 109523.

10. Fahy JV , Dickey BF. Airway mucus function and dysfunction. N Engl J Med 2010; 363:2233-47.

11. Proceedings of the A TS workshop on refractory asthma: current understanding, recommendations, and unanswered questions. American Thoracic Society. Am J Respir Crit Care Med 2000;162:2341-51.

12. McCall MN, Bolstad BM, Irizarry RA. Frozen robust multiarray analysis (fRMA).Biostatistics 2010;11:242-53.

13. Ritchie ME, Phipson B, Wu D, Hu Y , Law CW, Shi W, et al. Limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res 2015;43:e47.

14. Langfelder P , Horvath S. WGCNA: an R package for weighted correlation network analysis. BMC Bioinformatics 2008;9:559.

15. Kubysheva N, Boldina M, Eliseeva T, Soodaeva S, Klimanov I, Khaletskaya A, et al. Relationship of serum levels of IL-17, IL-18, TNF-alpha, and lung function parameters in patients with COPD, asthma–COPD overlap, and bronchial asthma.Mediators Inflamm 2020;2020:4652898.

16. ?Astrand AB, Hemmerling M, Root J, Wingren C, Pesic J, Johansson E, et al. Linking increased airway hydration, ciliary beating, and mucociliary clearance through ENaC inhibition. Am J Physiol Lung Cell Mol Physiol 2015;308:L22-32.

17. Goldfarbmuren KC, Jackson ND, Sajuthi SP, Dyjack N, Li KS, Rios CL, et al. Dissecting the cellular specificity of smoking effects and reconstructing lineages in the human airway epithelium. Nat Commun 2020;11:2485.

18. Schiller HB, Montoro DT, Simon LM, Rawlins EL, Meyer KB, Strunz M, et al.The human lung cell atlas: a high-resolution reference map of the human lung in health and disease. Am J Respir Cell Mol Biol 2019;61:31-41.

19. Wang S, Song R, Wang Z, Jing Z, Wang S, Ma J. S100A8/A9 in inflammation.Front Immunol 2018;9:1298.

20. Hinks TSC, Brown T, Lau LCK, Rupani H, Barber C, Elliott S, et al. Multidimensional endotyping in patients with severe asthma reveals inflammatory heterogeneity in matrix metalloproteinases and chitinase 3–like protein 1. J Allergy Clin Immunol 2016;138:61-75.

21. Moore WC, Hastie A T, Li X, Li H, Busse WW, Jarjour NN, et al. Sputum neutrophil counts are associated with more severe asthma phenotypes using cluster analysis. J Allergy Clin Immunol 2014;133:1557-63.e5.

22. Didon L, Zwick RK, Chao IW, Walters MS, Wang R, Hackett NR, et al. RFX3 modulation of FOXJ1 regulation of cilia genes in the human airway epithelium.Respir Res 2013;14:70.

23. Becker-Heck A, Zohn IE, Okabe N, Pollock A, Lenhart KB, Sullivan-Brown J, et al. The coiled-coil domain containing protein CCDC40 is essential for motile cilia function and left–right axis formation. Nat Genet 2011;43:79-84.

24. Pazour GJ, Agrin N, Walker BL, Witman GB. Identification of predicted human outer dynein arm genes: candidates for primary ciliary dyskinesia genes. J Med Genet 2006;43:62-73.

25. Ibanez-Tallon I, Gorokhova S, Heintz N. Loss of function of axonemal dynein Mdnah5 causes primary ciliary dyskinesia and hydrocephalus. Hum Mol Genet 2002;11:715-21.

26. Lechtreck KF, Delmotte P , Robinson ML, Sanderson MJ, Witman GB. Mutations in Hydin impair ciliary motility in mice. J Cell Biol 2008;180:633-43.

27. Bartel S, Schulz N, Alessandrini F, Schamberger AC, Pagel P , Theis FJ, et al. Pulmonary microRNA profiles identify involvement of Creb1 and Sec14l3 in bronchial epithelial changes in allergic asthma. Sci Rep 2017;7:46026.

28. Danahay H, Atherton H, Jones G, Bridges RJ, Poll CT. Interleukin-13 induces a hypersecretory ion transport phenotype in human bronchial epithelial cells. Am J Physiol Lung Cell Mol Physiol 2002;282:L226-36.

29. Gu H, Mickler EA, Cummings OW, Sandusky GE, Weber DJ, Gracon A, et al.Crosstalk between TGF-b1 and complement activation augments epithelial injury in pulmonary fibrosis. FASEB J 2014;28:4223-34.

30. Krane CM, Deng B, Mutyam V , McDonald CA, Pazdziorko S, Mason L, et al.Altered regulation of aquaporin gene expression in allergen and IL-13–induced mouse models of asthma. Cytokine 2009;46:111-8.

31. Kuperman DA, Huang X, Koth LL, Chang GH, Dolganov GM, Zhu Z, et al. Direct effects of interleukin-13 on epithelial cells cause airway hyperreactivity and mucus overproduction in asthma. Nat Med 2002;8:885-9.

32. Laitinen LA, Heino M, Laitinen A, Kava T, Haahtela T. Damage of the airway epithelium and bronchial reactivity in patients with asthma. Am Rev Respir Dis 1985;131:599-606.

33. Thomas B, Rutman A, Hirst RA, Haldar P , Wardlaw AJ, Bankart J, et al. Ciliary dysfunction and ultrastructural abnormalities are features of severe asthma.J Allergy Clin Immunol 2010;126:722-9.e2.

34. Jackson ND, Everman JL, Chioccioli M, Feriani L, Goldfarbmuren KC, Sajuthi SP, et al. Single-cell and population transcriptomics reveal pan-epithelial remodeling in type 2-high asthma. Cell Rep 2020;32:107872.

35. Lachowicz-Scroggins ME, Boushey HA, Finkbeiner WE, Widdicombe JH. Interleukin-13–induced mucous metaplasia increases susceptibility of human airway epithelium to rhinovirus infection. Am J Respir Cell Mol Biol 2010;43:652-61.

36. Gomperts BN, Kim LJ, Flaherty SA, Hackett BP . IL-13 regulates cilia loss and foxj1 expression in human airway epithelium. Am J Respir Cell Mol Biol 2007; 37:339-46.

37. Kang JH, Hwang SM, Chung IY . S100A8, S100A9 and S100A12 activate airway epithelial cells to produce MUC5AC via extracellular signal-regulated kinase and nuclear factor–kappaB pathways. Immunology 2015;144:79-90.

38. Zhao J, Endoh I, Hsu K, Tedla N, Endoh Y , Geczy CL. S100A8 modulates mast cell function and suppresses eosinophil migration in acute asthma. Antioxid Redox Signal 2011;14:1589-600.

39. Heikamp EB, Patel CH, Collins S, Waickman A, Oh MH, Sun IH, et al. The AGC kinase SGK1 regulates TH1 and TH2 differentiation downstream of the mTORC2 complex. Nat Immunol 2014;15:457-64.

40. Qin L, Gibson PG, Simpson JL, Baines KJ, McDonald VM, Wood LG, et al. Dysregulation of sputum columnar epithelial cells and products in distinct asthma phenotypes. Clin Exp Allergy 2019;49:1418-28.

41. Jones AC, Troy NM, White E, Hollams EM, Gout AM, Ling KM, et al. Persistent activation of interlinked type 2 airway epithelial gene networks in sputum-derived cells from aeroallergen-sensitized symptomatic asthmatics. Sci Rep 2018;8:1511.

42. Ramasamy A, Curjuric I, Coin LJ, Kumar A, McArdle WL, Imboden M, et al. A genome-wide meta-analysis of genetic variants associated with allergic rhinitis and grass sensitization and their interaction with birth order. J Allergy Clin Immunol 2011;128:996-1005.

43. Sugier PE, Brossard M, Sarnowski C, V aysse A, Morin A, Pain L, et al. A novel role for ciliary function in atopy: ADGRV1 and DNAH5 interactions. J Allergy Clin Immunol 2018;141:1659-67.e11.

44. Rossios C, Pavlidis S, Hoda U, Kuo CH, Wiegman C, Russell K, et al. Sputum transcriptomics reveal upregulation of IL-1 receptor family members in patients with severe asthma. J Allergy Clin Immunol 2018;141:560-70.

45. Madouri F, Guillou N, Fauconnier L, Marchiol T, Rouxel N, Chenuet P , et al.Caspase-1 activation by NLRP3 inflammasome dampens IL-33–dependent house dust mite–induced allergic lung inflammation. J Mol Cell Biol 2015;7: 351-65.

46. Stechschulte LA, Sanchez ER. FKBP51—a selective modulator of glucocorticoid and androgen sensitivity. Curr Opin Pharmacol 2011;11:332-7.

47. Vitellius G, Fagart J, Delemer B, Amazit L, Ramos N, Bouligand J, et al. Three novel heterozygous point mutations of NR3C1 causing glucocorticoid resistance.Hum Mutat 2016;37:794-803.

48. van Dijk EM, Menzen MH, Spanjer AI, Middag LD, Brandsma CA, Gosens R.Noncanonical WNT-5B signaling induces inflammatory responses in human lung fibroblasts. Am J Physiol Lung Cell Mol Physiol 2016;310:L1166-76.

49. Wan WY , Hollins F, Haste L, Woodman L, Hirst RA, Bolton S, et al. NADPH oxidase-4 overexpression is associated with epithelial ciliary dysfunction in neutrophilic asthma. Chest 2016;149:1445-59.

50. Bailey KL, Bonasera SJ, Wilderdyke M, Hanisch BW, Pavlik JA, DeVasure J, et al.Aging causes a slowing in ciliary beat frequency, mediated by PKCepsilon. Am J Physiol Lung Cell Mol Physiol 2014;306:L584-9.

51. Jakiela B, Gielicz A, Plutecka H, Hubalewska-Mazgaj M, Mastalerz L, Bochenek G, et al. Th2-type cytokine-induced mucus metaplasia decreases susceptibility of human bronchial epithelium to rhinovirus infection. Am J Respir Cell Mol Biol 2014;51:229-41.

52. Post S, Heijink IH, Hesse L, Koo HK, Shaheen F, Fouadi M, et al. Characterization of a lung epithelium specific E-cadherin knock-out model: implications for obstructive lung pathology. Sci Rep 2018;8:13275.

53. Vieira Braga FA, Kar G, Berg M, Carpaij OA, Polanski K, Simon LM, et al. A cellular census of human lungs identifies novel cell states in health and in asthma.Nat Med 2019;25:1153-63.

54. Hackett NR, Shaykhiev R, Walters MS, Wang R, Zwick RK, Ferris B, et al. The human airway epithelial basal cell transcriptome. PLoS One 2011;6:e18378.

Poole A, Urbanek C, Eng C, Schageman J, Jacobson S, O’Connor BP, et al. Dissecting childhood asthma with nasal transcriptomics distinguishes subphenotypes of disease. J Allergy Clin Immunol 2014;133:670-8.e12.