探討NLRP3發炎體及共生菌相在空氣懸浮微粒PM2.5引發的過敏氣道發炎中扮演的角色

Abstract

現今社會接觸汙染物的機率增加，令表皮屏障長期損傷造成共生菌相失調及持續的發炎反應，使整體過敏性發炎疾病盛行率升高。空氣懸浮微粒（particulate matter）PM2.5因其物理、化學特性及致病性引起公眾關注。已知PM2.5能夠藉由累積氧化壓力及引發偏向第二型發炎反應(type 2 inflammation)使過敏性呼吸道發炎症狀加劇。本研究將透過兩個面向來探討PM2.5在呼吸道過敏性發炎反應帶來的影響。第一部份，探討PM2.5造成的共生菌相失調。第二部份，探討PM2.5引起的氧化壓力是否活化細胞內的NLRP3發炎體(NLRP3 inflammasome)，並探討該訊息傳遞如何影響呼吸道中第二型發炎反應啟動。 藉由在雞卵白蛋白（ovalbumin；OVA）激發的小鼠氣喘模式中加入鼻腔給予PM2.5的環節，我們建立了兩種不同的小鼠模式。兩種模式差異在於是否添加鋁明礬（alum）佐劑，避免PM2.5與鋁明礬功能上有未知重複性。第一部份，採集小鼠糞便做共生菌相分析，並將結果合併血清中OVA特異性免疫球蛋白含量進行相關性檢定。第二部份，使用MCC950抑制NLRP3發炎體訊息傳遞，觀察是否改善動物模式中的呼吸道發炎反應。並在細胞實驗中，測量肺泡巨噬細胞（alveolar macrophages）、呼吸道上皮細胞株A549及LA-4以及嗜中性球（neutrophil）經PM2.5刺激後發炎體相關訊息傳遞的反應。 在兩種模式中，OVA致敏小鼠經過PM2.5暴露後都產生更劇烈的呼吸道反應，肺中CD4+ T細胞活化狀態也更加提升。在未加入鋁明礬的模式中，PM2.5暴露加劇OVA致敏小鼠肺中嗜酸性發炎（eosinophilic inflammation）及TSLP提升，引發嗜中性發炎（neutrophilic inflammation）及IL-17表現。OVA致敏小鼠在經過PM2.5暴露後，原本血清中上升的OVA特異性G2a型免疫球蛋白（immunoglobulin G2a；IgG2a）受到抑制。菌相分析中發現PM2.5及OVA刺激對小鼠的共生菌相產生不同的調節作用，此外血清中的OVA特異性免疫球蛋白含量與厭氧支原體屬(Anaeroplasma)及鼠桿狀菌科(Muribaculaceae)含量呈正相關，與乳桿菌屬(Lactobacillus)呈負相關。PM2.5¬單獨並未造成呼吸道症狀但仍會提升梭菌綱(Clostridia)中瘤胃菌科(Ruminococcaceae)和毛螺菌科(Lachnospiraceae)含量，並與免疫球蛋白呈負相關。另一方面，OVA致敏小鼠肺部NLRP3發炎體的表現會因PM2.5刺激而更加提升，MCC950顯著降低肺部免疫細胞浸潤，並降低肺部趨化因子eotaxin-1、CCL3及CCL5的表現量。細胞實驗中，我們發現NLRP3發炎體的訊號來自嗜中性球而非一般預測的肺泡巨噬細胞或呼吸道上皮細胞。實驗中，體外活化的嗜中性球在遭遇PM2.5或外加IL 1β刺激後，其功能性細胞激素TNF及趨化因子CCL3的分泌量都會顯著上升。 總結以上實驗結果，PM2.5透過提升呼吸道嗜酸性球及嗜中性球數量來加劇呼吸道過敏性發炎及呼吸道過度反應。在OVA致敏及未致敏小鼠中，PM2.5暴露表現出不同的共生菌相組成，此外由嗜中性球調控的NLRP3發炎體訊息傳遞在PM2.5在呼吸道過敏性發炎中對於提升肺部細胞浸潤扮演重要角色。此結果提升我們對於氣喘病因學的認知，顯示共生菌相具備成為氣喘生物性指標的潛力，並指出嗜中性球中的NLRP3發炎體活化可能影響疾病的嚴重程度。

Increased contact with pollutants in industrialization causes constant epithelial damage, followed by microbiota dysbiosis and chronic inflammation, leading to a burst in the prevalence of inflammatory diseases. Among all, particulate matter 2.5 (PM2.5) has drawn public concerns due to its physicochemical characteristics and pathological role. PM2.5 aggravates allergic airway inflammation by inducing type 2 immunity-biased response and excessive accumulation of oxidative stress. Thus, we are interested in two aspects; the first one lay on the PM2.5-induced microbiota dysbiosis; whereas the second one is on nucleotide-binding oligomerization domain leucine-rich repeat and pyrin domain containing 3 (NLRP3) inflammasome signaling in response to PM2.5-induced accumulation of oxidative stress since their role in the initiation of type 2 inflammation was not fully understood. Ovalbumin (OVA)-induced murine models with intranasal PM2.5 were applied to study the changes in features of airway allergic inflammation. Besides the common model with alum adjuvant, we included an alum-free model to prevent possible redundancy between PM2.5 and alum adjuvant. First, we analyzed the compositional shift of microbiota and their correlation to the serum level of OVA-specific immunoglobulins. Second, we applied the inflammasome inhibitor, MCC950, to the animal and evaluated the alterations in the allergic features. In vitro, we analyzed the inflammasome signaling of alveolar macrophages (AM), airway epithelial cell (AEC) line A549 and LA 4, and neutrophils after PM2.5 stimulation. In both models, PM2.5 aggravated airway hyperresponsiveness (AHR) and increased the activation status of lung-infiltrating CD4+ T cells in OVA-sensitized mice. In the alum free model, PM2.5 enhanced eosinophilic inflammation and TSLP expression; induced neutrophilic inflammation and IL-17 production in the lungs; and suppressed the sera OVA-specific IgG2a levels. We observed that PM2.5 and OVA immunization induced different dysbiosis in the gut. The serum level of OVA-specific immunoglobulins was positively correlated with the abundance of Anaeroplasma and Muribaculaceae; negatively correlated with Lactobacillus. Naïve mice exposed to PM2.5 did not develop AHR but still obtained an increased abundance of Ruminococcaceae and Lachnospiraceae. Both Clostridia are negatively correlated to the level of OVA-specific immunoglobulins. On the other hand, PM2.5 enhanced NLRP3 expression in the lungs of OVA-sensitized mice. Treatment by MCC950 suppressed the lung infiltrations with decreased expression of chemokines eotaxin-1, CCL3, and CCL5. Following in vitro experiments identified neutrophils instead of AM or AEC as the source of PM2.5-enhanced inflammasome signaling. Further, the addition of inflammasome product, IL 1β, enhanced TNFα and CCL3 production in neutrophils. Collectively, PM2.5 aggravated allergic airway inflammation by enhancing eosinophils and neutrophils in the lungs and exacerbated AHR. The particle generated a unique pattern of dysbiosis between naïve and OVA-sensitized mice. In addition, the study emphasized the role of neutrophil-derived inflammasome in the PM2.5-induced effect on allergic airway inflammation. The data collectively advance our knowledge on the etiology of asthma by suggesting microbiota as biomarkers and proposing a new controller, the inflammasome in neutrophils, in disease severity.