The Role of Macrophages in the Pathogenesis of Severe Asthma
- 주제(키워드) Asthma , autoimmunity , epithelial cells , extracellular traps , innate lymphoid cells , macrophages , monocytes , neutrophils , severe asthma
- 주제(DDC) 570
- 발행기관 아주대학교
- 지도교수 Hae-Sim Park
- 발행년도 2023
- 학위수여년월 2023. 8
- 학위명 박사
- 학과 및 전공 일반대학원 생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000032825
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Background: Severe asthma (SA) is often associated with neutrophilic inflammation, which is defined by neutrophil infiltration in the airway, resulting in resistance to current anti-asthmatic medication and a decline in lung function. Excessive amounts of neutrophil extracellular traps (NETs), which are released from activated neutrophils, harm airway epithelial cells (AECs) and activate eosinophils in severe asthmatic patients. The formation of NETs and the survival/migration of neutrophils are mainly controlled by a variety of pro-inflammatory cytokines (tumor necrosis factor-α, IL-1β, IL-8, and IL-6) derived mainly from activated M1 macrophages (M1Mφ) and classical monocytes (CMs). The literature emphasizes that a higher number of CMs/M1Mφ is noted in patients with neutrophilic asthma. Furthermore, CMs could release monocyte extracellular traps (MoETs); however, their functions in asthma pathophysiology remain unknown. Additionally, cytokeratin 19 (CK19) has been shown to be highly expressed in AECs. The presence of serum CK19-specific immunoglobulin (Ig)G autoantibodies in patients with toluene diisocyanate-induced asthma suggests that AEC damage occurs in one of the sub-phenotypes of asthma presenting a high incidence of SA and neutrophilic activation profiles. The triggers of CK19 release and their functions in neutrophilic inflammation in SA have not been investigated. Objective: This thesis has evaluated: 1) the mechanisms of how CMs/M1Mφ induce the activation of neutrophils/innate lymphoid cells (ILCs) in SA; and 2) the effects of CK19-specific IgG on the activations of neutrophils and CMs ex vivo and in vivo. Methods: In the first study, serum levels of monocyte chemoattractant protein-1 (MCP-1) and soluble suppression of tumorigenicity 2 (sST2) were measured in 39 patients with SA and 98 those with nonsevere asthma (NSA). CMs/Mφ were isolated from patients with SA (n = 19) and those with NSA (n = 18) and treated with LPS/interferon-gamma. MoETs/M1Mφ extracellular traps (M1ETs) were evaluated by western blotting, immunofluorescence, and PicoGreen assay. The effects of MoETs/M1ETs on neutrophils, AECs, ILC1, and ILC3 were assessed in vitro and in vivo. In the second study, we recruited 60 adult asthmatics (35 patients with eosinophilic and 25 those with noneosinophilic asthma) and 15 healthy controls (HCs) to evaluate autoimmune mechanisms in asthma. Various clinical parameters (including lung function, blood eosinophils, total IgE, and sputum eosinophil/neutrophil counts) were collected. The levels of myeloperoxidase (MPO) as well as autoantibodies (CK19-specific IgG and NET-specific IgG) in serum were measured using enzyme-linked immunoassay. In addition, the roles of CK19-specific IgG-derived immune complexes (ICs) to induce immune responses by the formation of NETs were investigated ex vivo. To find a causative factor inducing CK19 production, MoETs were tested in vivo. Results: The SA group had significantly higher CM counts as well as higher levels of serum MCP-1/sST2 than the NSA group. Moreover, the SA group had significantly greater production of MoETs/M1ETs (from CMs/M1Mφ) than the NSA group. The levels of MoETs/M1ETs were positively correlated with blood neutrophils and serum levels of MCP-1/sST2, but negatively correlated with FEV1%. In vitro/in vivo studies demonstrated that MoETs/M1ETs could activate AECs, neutrophils, ILC1, and ILC3 by increased migration as well as proinflammatory cytokine production. MoETs have been shown to be a causative factor to induce CK19 and CK19-specific IgG production. Furthermore, the levels of serum CK19-specific IgG were significantly higher in asthmatic patients than in HCs. Among patients with NEA, asthmatics with high levels of CK19-specific IgG had higher levels of MPO and NET-specific IgG than those without CK19-specific IgG (P = 0.020 and P = 0.017, respectively). Moreover, the ICs from asthmatics with high CK19-specific IgG could enhance the NET formation and reactive oxygen species production (neutrophil activation) which was suppressed by N-acetylcysteine and anti-CD16 antibody treatment. Conclusions: This thesis demonstrated the roles of CMs/M1Mφ in the airway inflammation of neutrophilic inflammation in SA. CMs/M1Mφ could enhance asthma severity mediated by the release of their ETs (MoETs and M1ETs), thereby inducing neutrophil recruitment and activation. Serum sST2 and MCP-1 could be potential biomarkers for predicting the presence of MoETs and M1ETs. Modulating MoETs and M1ETs by anti-IL-33 and ST2 antibodies could be new therapeutics for controlling SA where the steroid functions have limitations. Additionally, MoETs could induce the release of CK19 from AECs, which further facilitates the production of CK19-specific IgG and NET formation, contributing to the development of an autoimmune phenotype.
more초록/요약
연구배경: 중증 천식 (SA)은 기도내 호중구 염증을 동반하며, 이는 현재 사용되고 있는 천식 약제에 반응하지 않아, 폐 기능 감소를 동반한다. 기도에서 활성화된 호중구는 호중구 세포외 트랩 (NET)을 방출하여, 기도 상피세포(AEC)를 손상시키고, 이는 동시에 호산구를 활성화한다. NET의 형성과 호중구의 수명 연장과 이동 증가는 다양한 전염증성 사이토카인 (tumor necrosis factor-α, IL-1β, IL-8 및 IL-6)에 의해 매개되며, 여기에는 활성화된 M1 대식세포 (M1Mφ) 및 고전적 단핵구(CM)가 관여하며, CM은 monocyte extracellular traps (MoET)을 방출한다. 호중구성 천식 환자에서 CMs/M1Mφ의 증가를 보고한 논문도 있으나, 구체적인 역할과 기능은 규명되지 않았다. 또한 cytokeratin 19 (CK19)는 AEC에서 높게 발현되는 구조 단백질로, 톨루엔 디이소시아네이트에 의한 천식 환자의 혈청에서 CK19 특이 면역글로불린(IgG) 치의 증가가 보고된 바 있으며, 이는 AEC 손상, 호중구 활성화 및 천식의 중증도와의 관련성을 시사한다. 목적: 본 연구의 목적은 1) CMs/M1Mφ가 SA에서 호중구/선천성 면역구(ILCs)의 활성화를 유도하는 기전과, 2) MoET/M1ET 에 의한 자가 면역 기전을 규명하고자 하였다. 방법: 첫 번째 연구에서는, 39명의 SA 환자와 98명의 비 중증 천식(NSA) 환자로부터 혈청 monocyte chemoattractant protein-1 (MCP-1) 및 soluble suppression of tumorigenicity 2 (sST2) 수치를 측정하였다. CMs/Mφ는 SA 환자(n = 19)와 NSA 환자 (n = 18)에서 분리하였고 LPS/인터페론-감마를 처리하였다. MoETs/M1Mφ extracellular traps (M1ETs)은 웨스턴 블롯팅, 면역형광 및 PicoGreen 분석으로 평가하였다. 호중구, AEC, ILC1 및 ILC3에 대한 MoET/M1ET의 효과는 시험관 내 및 생체 내 실험을 통하여 평가하였다. 두 번째 연구에서는, 천식의 자가면역 기전을 평가하기 위해 성인 천식 환자 60명 (호산구성 천식 환자 35명, 비호산구성 천식 환자 25명)와 15명의 건강 대조군(n=15)을 모집하고, 다양한 임상 매개변수(폐 기능, 혈액 호산구, 총 IgE 및 객담 호산구/호중구 수 포함)를 수집하였다. 혈청 내 myeloperoxidase (MPO) 및 autoantibodies (CK19-specific IgG 및 NET- specific IgG)치는 효소면역측정법을 이용하여 측정하였다. 또한 CK19-specific IgG 유래 면역 복합체(IC)가 NET 형성을 통하여 면역 반응을 유도하는 기전을 생체내 실험을 통하여 관찰하였으며, AEC에서 MoET에 의한 CK19 생성 유도를 확인하였다. 결과: 혈청 MCP-1/sST2 수치는 NSA군보다 SA 군에서 유의하게 높았으며, CMs/M1Mφ로부터 MoETs/M1ETs 생성도 SA 군에서 NSA 군에 비해 유의하게 높았다. MoETs/M1ETs치는 혈중 호중구 및 MCP-1/sST2의 혈청 수치와 양의 상관관계가 있었고, FEV1%치와는 음의 상관관계가 관찰되었다. 시험관내/생체내 연구 결과, MoET/M1ET 처리는 AEC 활성화와 전염증성 사이토카인 생성이 증가하였고, 호중구/ILC1/ILC3 활성화/이동을 증가시켰다. MoET는 AEC 에서 CK19 및 CK19 특이적 IgG 생성을 유도하였고, 혈청 CK19 특이 IgG치는 천식 환자에서 HC 에 비해 유의하게 높았다. 비호산구성 천식 환자 중 CK19 특이 IgG 수치가 높은 천식 환자군은 낮은 군에 비해 혈청 MPO 및 NET 특이 IgG 치가 유의하게 높았다(각각 P = 0.020, P = 0.017). 또한, 높은 CK19-특이적 IgG를 가진 천식 환자에서 유래한 면역 복합체(IC)는 호중구 활성화(NE 및 활성 산소 생성)를 유도하였고, 이는 N-아세틸시스테인과 항-CD16 항체 치료에 의해 억제되었다. 결론: SA 환자에서, 대식구로부터 MoETs/M1ETs의 방출 증가는 호중구 활성화를 통하여 천식의 중증도를 악화시킨다. 혈청 sST2/MCP-1치는 MoET/M1ET 생성을 예측하기 위한 잠재적인 바이오마커 갸능성을 제시할 수 있고, 항 IL-33/ST2 항체는 MoET/M1ET에 의한 호중구성 기도염증을 억제하여, 치료제로서 역할 가능성도 제시한다. 또한 MoET는 AEC에서 CK-19의 방출과 CK19 특정 IgG의 생성을 유도하여, 자가 면역 기전에 의한 기도 염증을 증가시킨다.
more목차
CHAPTER Ⅰ 1
Contribution of monocyte and macrophage extracellular traps to neutrophilic airway inflammation in severe asthma 1
I. INTRODUCTION 2
II. MATERIALS AND METHODS 4
A. Materials 4
B. Study subjects 4
C. Protocol for isolation of immune cells 5
D. Human Mφ subset induction 9
E. The treatment protocols of human CMs and Mφ ex vivo 9
F. The treatment protocols of human blood neutrophils and AECs 10
G. The treatment protocols of human and mouse ILCs 11
H. Immunofluorescence assay 11
I. Migration, ROS, and apoptosis assays 12
J. Mouse experiments 12
K. Statistical analysis 14
III. RESULTS 15
A. Higher serum MCP-1 levels in patients with SA 15
B. Increased MoETs/M1ETs in the patients with SA 20
C. Signaling pathways of MoETs/M1ETs releases 28
D. The effects of MoETs/M1ETs on neutrophil activation in SA 33
E. The effects of MoETs/M1ETs on AECs in vitro 38
F. The effects of MoETs/M1ETs on human ILC activation 41
G. In vivo experiments in mice 45
H. The effects of MoETs/M1ETs in vivo 50
IV. DISCUSSION 55
CHAPTER II 62
Association between cytokeratin 19-specific IgG and neutrophil activation in asthma 62
I. INTRODUCTION 63
II. MATERIALS AND METHODS 65
A. Materials 65
B. Establishment of in vivo mouse models 65
C. Mouse ELISA 66
D. Lung tissue protein analysis 66
E. Lung neutrophil isolation and treatment in vivo 67
F. Study subjects and clinical parameters 67
G. Human ELISA for detecting serum CK19-IgG 68
H. Immunoblotting analysis 68
I. Immune cell isolation and ET formation 69
J. IgG and IC preparation 69
K. Evaluation of neutrophil activation 70
L. AEC culture and stimulation 70
M. Statistical analysis 71
III. RESULTS 72
A. MoET-induced CK19/CK19-specific IgG production in vivo 72
B. The effects of CD16/CD16.2 on neutrophilic inflammation in NA 72
C. Comparison of serum CK19-IgG level between EA and NEA 80
D. Circulating IgG involved in neutrophil degranulation 87
E. IC-induced NET formation mediated by CD16 receptor 93
F. Autocrine functions of ICs on monocyte activation in vitro 96
IV. DISCUSSION 99
CONCLUSIONS 105
