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성인 천식에서 혈청 CC16 수준의 임상적 유용성

Clinical Relevance of Serum CC16 in Adult Asthma

  • 정창규 (Chang-Gyu Jung, 아주대학교 일반대학원)

초록/요약

Introduction: Club cell 16-kDa secretory protein (CC16) is a lung surfactant protein secreted primarily by terminal and respiratory bronchioles, which levels can be altered by environmental stimuli. However, the role of CC16 in asthma has not yet been fully evaluated. Objective: To evaluate the clinical relevance of serum CC16 level in adult asthma, we evaluated CC16 level in the serum of adult asthmatics in terms of clinical and immunologic parameters in the real-world clinical setting of adult asthmatics, and analyzed the effect of CC16 on human airway epithelial cells (HAECs) through in vitro experiments. Methods: Sixty-three adult asthmatics who had maintained regular anti-asthmatic medication including inhaled corticosteroid (ICS), and 61 healthy controls (HCs) were recruited in Ajou University Hospital, Suwon, South Korea. The asthmatic patients were classified into two study groups according to changes in forced expiratory volume in 1 second (FEV1)% after airway reversibility test after inhalation of a short-acting beta 2 agonist; the reversible group (increase in FEV1% 12% and/or >200 mL) and the stable group (≤200 mL). The serum levels of epithelial mediators, including eosinophil-derived neurotoxin (EDN), myeloperoxidase (MPO), tissue inhibitor of metalloproteinases (TIMP)-1, matrix metalloproteinase (MMP)-9, transforming growth factor (TGF)-β1, periostin, interleukin (IL)-8, and folliculin, as well as CC16 were measured by enzyme-linked immunosorbent assay (ELISA), and compared between the two groups. The associations between serum CC16 level and clinical/laboratory parameters were evaluated. Receiver operating characteristic (ROC) curve and multivariate logistic regression analyses were performed to evaluate the diagnostic valuate of serum CC16 in asthmatics. In vitro experiments were conducted to evaluate the effect of Dermatophagoides pteronyssinus antigen 1 (Der p1) on the production of CC16, tight-junction proteins (claudin-1, E-cadherin), epithelial cytokines (IL-8, MMP-9, TIMP-1), antioxidant biomarkers (heme oxygenase [HO]-1, nuclear factor erythroid 2–related factor [Nrf]-2, glutathione [GSH]), and reactive oxygen species (ROS) in a time-dependent manner. In addition, the effects of CC16 protein treatment on airway inflammation/remodeling, and oxidative stress were evaluated compared to dexamethasone treatment. Results: Serum CC16 levels showed significantly lower in the reversible group than in the stable group (467.7 ± 125.0 ng/mL vs. 616.7 ± 196.6 ng/mL, P = 0.005), although significantly higher levels of serum MPO (560.9 ± 234.0 ng/mL vs. 412.3 ± 242.8 ng/mL, P = 0.022)/MMP-9 (507.8 ± 259.5 ng/mL vs. 352.2 ± 240.9 ng/mL, P = 0.019) were noted in the reversible group than in the stable group. No differences were noted in levels of serum EDN, TIMP-1, TGF-β1, periostin, IL-8, or folliculin. In addition, higher fractional exhaled nitric oxide levels (49.3 ± 42.4 vs. 30.6 ± 32.9 ppb, P = 0.042) as well as lower baseline FEV1% (85.7% ± 10.7% vs. 92.6% ± 18.2%, P = 0.030)/maximal mid-expiratory flow% (55.8% ± 25.6% vs. 73.9% ± 27.7%, P = 0.025) were noted in the reversible group than in the stable group. A significant positive correlation was observed between serum CC16 level and FEV1% value in asthmatic subjects (r = 0.352, P = 0.005), and the ROC analysis demonstrated that cutoff value of serum CC16 level (below 496.0 ng/mL) could discriminate the reversible group of asthma from the stable group (area under the curve = 0.740, P = 0.004, sensitivity 64.7%, specificity 76.1%). Moreover, multiple logistic regression analysis revealed that serum CC16 level is the only independent predictor for airway reversibility in asthmatic subjects (odds ratio = 0.994, P = 0.008). Our in vitro study demonstrated that in HAECs, the expressions of CC16, claudin-1, and E-cadherin was significantly decreased and those of fibronectin, IL-8, MMP-9, and TIMP-1 were significantly increased in response to chronic Der p1 exposure. CC16 treatment (but not dexamethasone treatment) improved the expression of epithelial tight junctional proteins, including claudin-1/E-cadherin, and markedly reduced the production of IL-8, MMP-9, and TIMP-1, as well as the expression of fibronectin in Der p1-stimulated HAECs. Moreover, overproduction of ROS as well as reduction in HO-1, Nrf2, and GSH were observed in Der p1-stimulated HAECs, which was restored by CC16 treatment. In addition, significant attenuation of CC16 production in HAECs was noted in response to higher levels of oxidative stress induced by neutrophil and/or eosinophil extracellular traps. Conclusion: These findings suggest that decreased CC16 in asthmatic patients may contribute to chronic persistent airway inflammation followed by airway remodeling, resulting in reversible airway obstruction and lung function decline. Serum CC16 may be a potential biomarker for predicting airway reversibility and provide an additional therapeutic target for adult asthmatics who are resistant to current anti-asthmatic medications.

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초록/요약

서론: 클럽 세포 16-kDa 분비 단백질(CC16)은 말단 및 호흡 세기관지 상피 세포에서 분비되는 계면활성제 단백질이다. CC16의 수준은 환경 자극에 의해 변동 가능하나, 주된 만성 기도 질환인 기관지 천식에서 그 역할에 대한 규명이 필요하다. 연구목적: 본 연구의 목적은 성인 천식환자의 혈청 CC16 수준과 임상/면역학적 매개변수와의 관련성을 평가하고, 시험관내 실험을 통해 인간 기도 상피 세포들(HAECs)에 대한 CC16의 효과를 분석하여, 그 임상적 유용성을 평가하고자 하였다. 연구방법: 아주대학교병원 알레르기 내과에서 정기적으로 천식 치료를 받고 있는 19세 이상 성인 천식 환자 63명과 건강한 대조군 61명을 모집하여, 다양한 임상 매개변수를 측정하였다. 천식 환자들의 경우 기본 폐기능(1초 동안 강제 호기량[FEV1], 최고중간호기유속[MMEF] 포함), 분획날숨산화질소(FeNO) 검사와 함께, 기도 가역성 검사를 진행하였다. 기도 확장제 흡입 후 FEV1%의 변화에 따라 가역군(FEV1%가 12% 그리고/또는 200 mL이상 증가)과 안정군(200 mL 미만)으로 분류하였고, 혈청 내 CC16과 상피 세포 유래 사이토 카인(matrix metalloproteinase [MMP]-9, tissue inhibitor of metalloproteinases [TIMP]-1, transforming growth factor [TGF]-β1 등)과 염증 세포 유래 매개체(myeloperoxidase [MPO], eosinophil-derived neurotoxin [EDN] 등) 농도를 효소결합면역흡착측정(ELISA)으로 측정하고 비교하였다. 시험관내 연구로 Dermatophagoides pteronyssinus 항원 1(Der p1)의 노출에 따른 HAECs에서의 CC16, 치밀-이음 단백질(claudin-1, E-cadherin), 상피 세포 유래 사이토카인(Interleukin [IL]-8, MMP-9, TIMP-1), 항산화 생물표지자(heme oxygenase [HO]-1, nuclear factor erythroid 2–related factor [Nrf]-2, 환원 글루타싸이온[GSH]), 반응산소종(ROS)들의 생성에 대하여 평가하였고, 덱사메타손 치료와 비교하여, 기도 염증, 기도 재형성 및 산화 스트레스에 대한 CC16 단백질의 효능을 평가하였다. 연구결과: 전체 63명의 천식 환자 중에 가역군은 17명(27.0%), 안정군은 46명(73.0%) 이었다. 가역군이 안정군보다 혈청 CC16 수준이 유의하게 낮았고(467.7 ± 125.0 ng/mL vs. 616.7 ± 196.6 ng/mL, P = 0.005), 혈청 MPO (560.9 ± 234.0 ng/mL vs. 412.3 ± 242.8 ng/mL, P = 0.022)와 MMP-9 (507.8 ± 259.5 ng/mL vs. 352.2 ± 240.9 ng/mL, P = 0.019) 수준은 유의하게 높았다. FeNO는 가역군이 안정군에 비해 유의하게 높았고(49.3 ± 42.4 vs. 30.6 ± 32.9 ppb, P = 0.042), FEV1% (85.7% ± 10.7% vs. 92.6% ± 18.2%, P = 0.03), MMEF (55.8% ± 25.6% vs. 73.9% ± 27.7%, P = 0.025)는 가역군이 안전군에 비해 유의하게 낮았다. 또한 혈청 CC16 수준은 FEV1%과 유의한 양의 상관관계가 있었고(r = 0.352, P = 0.005), 천식 환자에서 기도 가역성을 예측할 수 있는 혈청 CC16 수준은 cut-off point: 496.0ng/mL 미만(area under the curve = 0.740, P = 0.004)에서 민감도 64.7%, 특이도 76.1%였다. 추가로, 다중 로지스틱 회귀 분석을 통해 CC16의 혈청 수준이 천식 환자에서 기도 가역성의 유일한 독립적 예측인자임을 확인하였다(odds ratio = 0.994 P = 0.008). 시험관내 연구에서는 Der p1에 만성적으로 노출된 HAECs에서 CC16, claudin-1 및 E-cadherin의 발현이 유의하게 감소되었고, fibronectin, IL-8, MMP-9 및 TIMP-1의 발현이 유의하게 증가된 것을 확인하였다. 그리고 CC16 치료는 Der p1에 의해 자극된 HAECs에서 claudin-1, E-cadherin의 발현을 유의하게 개선시켰고, IL-8, MMP-9, TIMP-1 및 fibronectin의 발현을 유의하게 감소시켰다. 더욱이 Der p1에 의해 자극된 HAECs에서 관찰되었던 ROS의 과잉 생산과 HO-1, Nrf2 및 GSH의 감소가 CC16 처리에 의해 유의하게 회복되었다. 결론: 성인 천식 환자에서 혈청 CC16 수준의 감소는 기도 가역성, 기도 염증 및 기도 재형성의 지속 및 폐 기능 악화에 기여하고, CC16 치료는 기도 가역성을 예측하는 생체지표로 이용 가능성과, 기도 가역성을 지닌 천식 환자를 위한 치료 타겟의 가능성을 제시하였다.

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목차

I. INTRODUCTION 1
A. Unmet needs in asthma 1
B. The role of epithelial cells in asthma 2
C. Reactive oxygen species (ROS) in asthma 3
D. Club cell 16-kDa secretory protein (CC16) 4
E. Aims of the thesis 5
II. MATERIALS AND METHODS 6
A. Study subjects and clinical evaluation 6
B. In vitro experiments 8
C. Statistical analysis 10
III. RESULTS 11
A. Study subjects and clinical/laboratory characteristics 11
B. In vitro experiments 13
IV. DISCUSSION 16
A. Serum CC16 level is a predictor for airway reversibility and lower lung function 16
B. CC16 as a therapeutic target for asthma 17
C. Anti-inflammatory and anti-oxidant effects of CC16 on HAECs 18
D. CC16 as a diagnostic biomarker for asthma 19
E. Limitations of the study 20
V. CONCLUSION 21
VI. REFERENCES 22
제2언어초록 48

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