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The Roles of Serum Amyloid A-1 and Tissue Inhibitor of Metalloproteinase-1 in Asthma Pathogenesis

초록/요약

Background: Neutrophilia (sputum neutrophils  65%) in the sputum and its mediators are attributed to neutrophilic airway inflammation that is associated with poor steroid responsiveness and severe asthma (SA). Although many researchers have investigated its underlying mechanisms, potential biomarkers and therapeutics, it has not been completely understood. Serum amyloid A (SAA) was proposed as a biomarker for neutrophilic asthma (NA) and was associated with impaired lung function. SAA1, a primary precursor of SAA, was involved in lipid metabolism, bacterial clearance and inflammatory regulations; nevertheless, its clinical significance and function in NA have not been studied. Furthermore, the presence of eosinophilia in the blood/sputum of severe asthmatic patients (type 2 SA) contributed to recurrent asthma exacerbations and low lung function even on maintenance medication including inhaled corticosteroids (ICS) and long-acting beta-agonist (LABA). In addition, tissue inhibitor of metalloproteinase-1 (TIMP1) is a member of the TIMP family and play the role of a major inhibitor of metalloproteinase-9 (MMP9) in extracellular tissues. The pathogenic mechanism of TIMP1 on airway inflammation and remodeling remained uncertain, but accumulating evidence has shown that TIMP1 and MMP9 co-work in airways to upregulate tissue remodeling. Objective: This thesis sought to investigate: 1) clinical relevance of serum SAA1 according to asthma phenotype (NA vs non-NA); 2) SAA1 production from airway epithelial cells (AECs) upon external/internal stimuli; 3) the effect of SAA1 on the activation of neutrophils/macrophages; 4) SAA1 expression in different asthma mouse models; 5) clinical relevance of serum TIMP1 level according to SA phenotype (type 2 SA vs non-type 2 SA); 6) TIMP1 production from AECs upon various stimuli; 7) the effect of TIMP1 on the activation of eosinophils/macrophages; and 8) the effects of TIMP1 and anti-TIMP1 antibody on airway inflammation and remodeling in vivo. Methods: In the first study, 122 adult asthmatics (78 patients with NA and 44 those with non-NA) and 60 healthy controls (HCs) were enrolled to measure serum SAA1 levels. The production of SAA1 from AECs and its effects on neutrophils and macrophages were investigated. In the second study, we enrolled 250 adult asthmatics (54 patients with SA and 196 those with non-SA) and 140 HCs to measure serum TIMP1 levels. The release of TIMP1 from AECs and its effects on the activations of eosinophils and macrophages were evaluated. Results: Serum SAA1 levels were significantly higher in asthmatic patients than in HCs (P = 0.014); among asthmatics, patients with NA showed significantly higher SAA1 levels than those with non-NA (P < 0.001). In vitro investigations showed that poly I-C stimulation markedly enhanced the production of SAA1 from AECs, which was further augmented by neutrophils; SAA1 could induce the production of IL6, IL8 and S100 calcium-binding protein A9 from AECs. Additionally, SAA1 activated peripheral neutrophils and monocyte-derived macrophages from asthmatics to induce the neutrophil extracellular trap (NET) formation and pro-inflammatory cytokine production. In ovalbumin (OVA)-induced allergic asthma mice, poly I-C stimulation significantly increased SAA1, IL17A, interferon-gamma and IL33 levels in the bronchoalveolar lavage fluid (BALF). The highest levels of SAA1 were noted in the BALF and sera of the NA mouse model, followed by the mixed granulocytic asthma model. Especially, SAA1 induced IL17 and retinoic acid receptor-related orphan receptor gamma t expressions from activated CD4+ T lymphocytes in OVA-induced allergic asthma mice. In the second study, significantly higher levels of serum TIMP1 were noted in asthmatics than in HCs, in the SA group than in the non-SA group and in the type 2 SA group than in the non-type 2 SA group (P < 0.01 for all). A negative correlation between serum TIMP1 and FEV1% values (r = –0.400, P = 0.003) was noted in the SA group. In vitro investigations demonstrated that TIMP1 was released from AECs in response to poly I-C, IL13, eosinophil extracellular traps (EET) and in coculture with eosinophils. TIMP1-stimulated mice showed eosinophilic airway inflammation, which was not completely suppressed by steroid treatment. In vitro and in vivo functional investigations showed that TIMP1 directly activated eosinophils and macrophages, and induced the release of EET and macrophages polarization toward M2 subset, which was suppressed by anti-TIMP1 antibody. Conclusions: These findings suggest that SAA1 may cause neutrophilic airway inflammation in adult asthmatic patients by activating neutrophils to form NET, macrophages to polarize toward the M1 subset, and CD4+ T cells to polarize toward T helper (Th) 2/Th17 predominant cells, resulting in steroid resistance. We propose SAA1 as the potential biomarker for the prediction of NA phenotype. In patients with SA, TIMP1 may contribute to the upregulation of eosinophilic airway inflammation and remodeling via migrating eosinophil into the airways and subsequently activating eosinophils to release EET, which further activate group 2 innate lymphoid cells. In the chronic allergic asthma mouse model, blocking TIMP1 with neutralizing antibodies reduced airway hyperresponsiveness, eosinophilic airway inflammation and remodeling. Taken together, modulating TIMP1 and EET production may be a potential treatment strategy for type 2 SA.

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초록/요약

배경: 천식은 가역성 기도 폐쇄를 특징으로 하는 만성 기도 염증성 질환이다. 이 중 5%-10%를 차지하는 중증 천식(SA)은 지속적인 기도 염증을 나타내며, ICS를 포함한 약물 치료에도 폐기능이 감소한다. Serum amyloid A (SAA)은 호중구성 천식(NA, 가래 호중구 수 65%)의 바이오마커 중 하나로 1초간 강제 호기량 감소 (FEV1%) 감소와 관련성이 보고되었다. 반면, tissue inhibitor of metalloproteinase-1 (TIMP1)은 TIMP 계열이며, 세포외 조직에서 주로 matrix metalloproteinases-9 (MMP9)의 억제제로 알려져 있고, 최근 TIMP1와 MMP9는 상호 작용을 통하여 기도 개형을 증가시킨다. SAA1과 TIMP1는 주로 기도 상피 세포 (AECs)에서 다양한 자극에 의해 분비되며, SA의 중증도에 영향을 미친다. 목적: 본 연구는 (1) 천식 표현형(NA vs non-NA)에 따른 혈청 SAA1의 임상적 유용성, (2) 외부/내부 자극에 의한AEC로부터 SAA1 생성, (3) 호중구/대식세포의 활성화에 대한 SAA1의 효과, (4) 천식 마우스 모델에서 SAA1의 효과, (5) SA 표현형(2형 대 비 2형)에 따른 임상적 매개변수와 관련한 혈청 TIMP1의 임상적 유의성, (6) 다양한 자극에 의한 AECs의 TIMP1 생성, (7) 호산구/대식세포의 활성에 의한 TIMP1의 효과, (8) TIMP1 및 항 TIMP1 항체가 생체 내 기도 염증/개형에 미치는 영향, 총 8가지를 확인하고자 하였다. 재료 및 방법: 첫번째 연구에서는, 122명의 천식 환자와 60명의 건강한 대조군(HCs)에서 혈청 SAA1를 측정하였다. 그리고 AECs에서 SAA1의 생산과 대식세포 및 호중구에 미치는 영향을 시험관 내 및 천식 마우스 모델에서 관찰하였다. 두 번째 연구에서는, 성인 천식 환자 250명(SA군, 54명; non-SA군, 196명)과 140명의 HCs 에서 혈청 TIMP1치를 측정하였다. 호산구 및 대식세포의 활성화에 대한 TIMP1의 효과와 AECs로부터 다양한 외부 자극에 따른 TIMP1의 방출 정도를 시험관내 및 생체내에서 평가하였다. 결과: 혈청 SAA1 수치는 천식 환자에서 HCs보다 유의하게 높았고(P = 0.014), 천식 환자들 중 호중구성 천식 환자군이 비 호산구성 천식군보다 유의하게 높았다 (P < 0.001). Poly I-C 처리 후 AECs에서 SAA1 생산이 현저하게 증가하였으며, 이는 호중구 처리 후 더욱 증가되었다. SAA1 처리 후, AECs에서 IL6, IL8 및 S100 칼슘 결합 단백질 A9 유리가 증가하였다. 또한, SAA1은 호중구에서 천식 환자의 말초 혈액에서 호중구 및 대식세포를 활성화하고, 호중구 세포외 트랩 형성 및 염증성 사이토카인 생산을 증가시켰다. 알부민으로 유발한 천식 마우스에서 poly I-C 자극은 기관지 폐포 세척액(BALF)에서 SAA1 수치과 IL17A/인터페론-감마/IL33 수치을 유의하게 증가시켰다. NA 마우스 모델의 BALF와 혈청에서 가장 높은 수치의 SAA1과 호중구 증가증이 나타났고, 혼합 과립구성 천식 마우스 모델이 그 뒤를 이었다. 특히, SAA1은 천식 마우스의 활성화된 CD4+ T 림프구로부터 RORt 발현을 유도하였다. 두 번째 연구에서, 혈청 TIMP1 수치는 천식 환자에서 HCs에 비해 유의하게 높았으며, 천식 환자중에서, non-SA군보다 SA군에서, 비2형군보다 2형 SA군에서 유의하게 높았다(각 P <0.01). SA 환자군에서 혈청 TIMP1과 FEV1 % 값 사이의 음의 상관관계(r = -0.400, P = 0.003)가 관찰되었다. 시험관 내 연구결과로 TIMP1는 AECs에서 다양한 외부 자극인 poly I-C, IL13, 호산구 세포외 트랩(EET) 및 호산구와 공동 배양 후 방출되었다. 동물 모델에서는 TIMP1 자극 후 호산구성 기도 염증이 관찰되었으며, 이는 스테로이드 치료로 완전히 억제되지 않았다. 또한, TIMP1은 호산구와 대식세포를 직접 활성화하고, EET와 대식세포의 방출을 유도하여, 항-TIMP1 항체 치료후 M2로의 분극화가 억제되었다. 결론: 이러한 결과는 SAA1이 NET 형성, M1 방향 대식세포 활성화 및 T 헬퍼(Th)2/Th17 우세 세포와 함께 호중구를 활성화 및 호중구 기도 염증을 유발하여, NA 또는 MA의 표현형에 기여한다. TIMP1은 SA 환자에서 호산구성 기도 염증을 강화하며, 혈청 TIMP1은 2형 SA에 대한 잠재적인 바이오마커 및/또는 치료 표적이 될 수 있다.

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목차

CHAPTER-I 1
Serum Amyloid A-1: A Biomarker for Neutrophilic Airway Inflammation in Adult Asthmatic Patients 1
I. INTRODUCTION 2
II. MATERIALS AND METHODS 4
A. Study subjects 4
B. Human AEC culture and stimulation 4
C. Peripheral blood neutrophil (PBN) and monocyte isolation and culture 5
D. Neutrophil extracellular trap (NET) induction 5
E. Reactive oxygen species (ROS) quantification 6
F. Neutrophil migration assay 6
G. Macrophage polarization and stimulation 6
H. Asthma mouse models 7
I. Measurement of airway hyperresponsiveness (AHR) 8
J. Differential cell count 9
K. Murine CD4+ T cell isolation and stimulation 9
L. Human T lymphocyte cell line culture and stimulation 9
M. ELISA 9
N. Western blot analysis 10
O. Immunofluorescence with confocal microscope 10
P. Statistical analysis 10
III. RESULTS 12
A. Clinical characteristics of the study subjects 12
B. Factors for inducing SAA1 production from AECs 16
C. Effect of SAA1 on neutrophil activation 18
D. Effect of SAA1 on macrophage activation 20
E. SAA1 expression in allergic asthma mice infected by virus 22
F. SAA1 expression in the mouse model of asthma phenotypes 24
G. Effect of SAA1 on Th2/Th17 cytokine expression from splenic CD4+ T cells of allergic asthma mice 26
H. Effect of SAA1 on Th2 cytokine releases from Jurkat T cell line 27
IV. DISCUSSION 28
CHAPTER-II 32
Tissue Inhibitor of Metalloproteinase-1 Enhances Eosinophilic Airway Inflammation in Severe Asthma 32
I. INTRODUCTION 33
II. MATERIALS AND METHODS 35
A. Study subjects 35
B. Human AEC culture and stimulation 35
C. Peripheral blood eosinophil (PBE) and monocyte stimulation 36
D. Eosinophil migration assay 37
E. Human mast cell line culture and stimulation 37
F. Human macrophage culture and stimulation 38
G. Mouse model 38
H. ELISA 40
I. Western blot analysis 40
J. Flow cytometry 40
K. Immunofluorescence with confocal microscope 42
L. Statistical analysis 43
III. RESULTS 44
A. Clinical characteristics and serum cytokine levels of the study subjects 44
B. Clinical characteristics of the asthmatic subjects according to serum TIMP1 level 48
C. TIMP1 and MMP9 production from human AECs 51
D. Effect of TIMP1 on eosinophil migration and activation 54
E. Effect of TIMP1 on LAD2 cells 56
F. Effect of TIMP1 on human macrophages 57
G. Effect of TIMP1 on airway inflammation in vivo 59
H. Effect of anti-TIMP1 antibody on airway inflammation of chronic allergic asthma mouse model 62
IV. DISCUSSION 66
CONCLUSION 69

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