Wide-target metabolome analysis to evaluate the impact of Mycobacterium abscessus infection to an in vitro airway epithelial model

다국어 초록 (Multilingual Abstract)

The prevalence of non-tuberculosis mycobacteria pulmonary disease (NTM-PD) is rising worldwide, especially in those with lung diseases, such as chronic obstructive pulmonary disease (COPD). Over the past decade, the number of NTM-PD patients in Korea and Japan has increased five-fold, and the five-year mortality rate for NTM-PD patients in Asia is approximately 30%. Despite the high burden of NTM-PD, the treatment process is not optimized. According to international guidelines, even if sputum cultures are negative, an additional 12 months of antibiotic treatment is recommended. This is because there are no appropriate criteria for determining the timing of a cure. The absence of biomarkers indicative of NTM-PD makes effective diagnosis, optimization of the treatment process, and prognosis assessment challenging. Clinical studies and in vitro studies have been conducted to identify biomarkers for NTM-PD.
However, clinical studies are not suitable for high-throughput screening and face difficulties in identifying biomarkers that indicate the early stages of infection in NTM-PD patients. Therefore, it is essential first to identify potential biomarker candidates through high-throughput screening and an in vitro airway epithelial model that can represent the early stages of infection. In vitro studies have predominantly used the bronchial airway instead of the actual infection site, the small airway. However, due to the increasing recognition of differences in cell composition and mucus secretion between the bronchial and small airways, research using the small airway, the actual infection site, is required. Additionally, metabolomic analysis related to NTM-PD has never been conducted in an in vitro airway epithelial model.
The effects of Mycobacterium abscessus subsp. abscessus (MAB) infection were evaluated using metabolomic analysis in vitro airway epithelial model. An in vitro airway epithelial model was used to evaluate the effects of MAB infection on normal primary human bronchial epithelial cells (normal HBECs) as a traditional in vitro airway epithelial model and COPD primary human small airway epithelial cells (COPD HSAECs) through metabolomic analysis. In normal HBECs, metabolites related to nitric oxide (NO) production and oxidative stress were identified. In COPD HSAECs, the vitamin B family which is essential for the survival and virulence of mycobacteria was also identified in addition to those related to NO production and oxidative stress. Additionally, pathway analysis revealed that arginine and proline metabolisms and urea cycle are closely related to MAB infection. The application of metabolomic research in vitro airway epithelial model provides insights into the host-pathogen interactions of NTM-PD and is expected to support studies identifying biomarkers for the diagnosis, optimization of the treatment, and prognosis of NTM-PD.

국문 초록 (Abstract)

만성 폐쇄성 폐질환(COPD) 환자와 같은 만성 폐질환을 가진 위험군에서 비결핵 항산균 폐질환(NTM-PD)은 국제보건의 중요한 문제로 간주되고 있다. 아시아에서 NTM-PD 진단 후 5년 내 사망률은 30%이며, 한국과 일본에서 NTM-PD를 진단받은 환자는 지난 10년간 5배 증가하였다. NTM-PD는 높은 위험도를 나타내지만 치료과정이 표준화되어 있지 않다. 국제가이드라인은 객담 배양이 음성으로 전환된 후 12개월간 추가적인 항생제 치료를 권장하고 있다. 이러한 추가적인 치료는 완치에 대한 명확한 기준이 없기 때문이다. NTM-PD 특이적 바이오마커의 부재로 인해 진단과 치료 표준화 및 예후를 판단하는 것은 어렵다.
NTM-PD 특이적 바이오마커 식별 연구는 임상 연구와 in vitro연구에서 수행되어 왔다. 하지만 임상 연구는 바이오마커 식별을 위한 high-throughput screening을 수행하기에 샘플 표본이 적고, NTM-PD환자들에게서 초기상태를 표지할 수 있는 바이오마커를 식별하기에 어려움이 있다. 그러므로 in vitro airway epithelial model을 이용한 연구가 high-throughput screening과 NTM감염 초기를 나타낼 수 있는 바이오마커 후보 물질을 식별할 수 있기 때문에 선행이 되어야 한다. In vitro 연구는 실제 감염 부위인 small airway아닌 bronchial airway가 주로 사용되어 왔다. 하지만 bronchial airway와 small airway간에 세포 구성 및 분비되는 mucus의 차이 때문에 실제 감염 부위인 small airway를 이용한 연구가 필요하다. 또한, in vitro airway epithelial model에서 NTM감염과 관련된 대사체 분석은 보고된 적이 없었다. 이 연구에서는 in vitro airway epithelial model에서 NTM에 속하는 기회 병원균인 Mycobacterium abscessus subsp. abscessus (MAB)감염에 의한 대사체 변화를 분석하여 NTM-PD 특이적인 바이오마커 식별을 위한 대사체학 연구 기반을 제공하고자 하였다.
기존 in vitro airway epithelial model인 normal primary human bronchial epithelial cells (normal HBECs)와 추가적으로 COPD primary human small airway epithelial cells (COPD HSAECs)에서 MAB감염에 의한 대사체 변화를 분석하였다. 공통적으로 산화 질소생성 및 산화스트레스와 관련된 대사체가 식별이 되었고, COPD HSAECs에서는 mycobacteria의 생존과 독성에 필수라고 알려져 있는 vitamin B family가 추가적으로 식별되었다. pathway analysis는 arginine and proline metabolism이 airway epithelial model에서 MAB감염과 깊은 관련이 있는 것으로 나타났다. 이 연구는 in vitro airway epithelial model에서 MAB감염과 관련이 있는 대사체를 식별하였다. In vitro airway epithelial model에서 대사체 분석은 host-pathogen interaction에관한 연구와 진단, 치료 표준화 및 예후를 위한 질환 특이적 바이오마커 식별 연구를 뒷받침하는 근거가 될 수 있을 것으로 기대한다.

목차 (Table of Contents)

• CHAPTER 1. INTRODUCTION 1
• CHAPTER 2. MATERIALS AND METHODS 10
• 2.1 Bacterial culture 10
• 2.2 Preparation of mouse feeder cells 11
• 2.3 Preparation of HAECs in air-liquid interface culture for differentiation 12
• 2.4 Infection protocol 16
• 2.5 Bacterial viability test 17
• 2.6 Immunocytochemistry (ICC)-stain 18
• 2.7 Extraction of metabolites 19
• 2.8 Pretreatment of hydrophilic metabolites 22
• 2.9 Pretreatment of phospholipid for profiling 22
• 2.10 Hydrophilic metabolite analysis 23
• 2.11 Phospholipid analysis 24
• 2.12 Peak processing and relative quantification of each compound 24
• 2.13 Statistical analysis of metabolites 25
• CHAPTER 3. RESULTS 27
• 3.1 Differentiation of HAECs cultured at the air-liquid interface 27
• 3.2 Infection of human airway epithelial cells with MAB 29
• 3.3 Evaluation of bactericidal effects of NE on MAB was estimated for bacterial viability test 29
• 3.4 Targeted LC-MS/MS analysis 31
• 3.4.1 Metabolite changes induced by NE in MAB 31
• 3.4.2 Metabolite changes induced by NE in HAECs infected with MAB 33
• 3.4.3 Metabolite changes in normal HBECs due to MAB infection 36
• 3.4.4 Metabolite changes in COPD HSAECs due to MAB infection 44
• 3.5 HAECs exhibit diverse metabolic changes by MAB infection 64
• CHAPTER 4. DISCUSSION 67
• CHAPTER 5. REFERENCES 71
• ABSTRACT IN KOREAN 76