β-羥基丁酸酯對過敏性免疫反應的影響

Abstract

β-羥基丁酸酯為一種由脂肪酸代謝而來的產物，也是在哺乳類動物當中最豐富的酮體，可以當作體內ATP生產的替代來源。在一般正常生理下，β-羥基丁酸酯的濃度非常低，然而在節食或是攝取生酮飲食的情況下，它的濃度能上升至2 mM。先前的研究指出在神經疾病的動物模式當中，β-羥基丁酸酯具有防止疾病惡化以及促進細胞再生的作用；也已經有研究證實提升血清中β-羥基丁酸酯的濃度可能有助於緩解過敏症狀，然而其中詳細的機制還有待更進一步的研究。因此本篇的研究目的主要是想探討β-羥基丁酸酯在過敏免疫反應當中所扮演的角色。在動物實驗中，我經由皮下包埋植入式滲透壓膠囊，長時間給予β-羥基丁酸酯並額外以腹腔注射提高體內的濃度後，在介白素-33所引起的肺部發炎小鼠模式當中，可以看到浸潤至肺部氣道腔室中的嗜酸性白血球數量有顯著的降低。根據細胞流式儀的實驗結果顯示，給予外源性β-羥基丁酸酯能夠抑制表現IL-5以及IL-13的第二型先天性淋巴細胞 (Group 2 Innate lymphoid cells; ILC2) 族群。接著，為了延長β-羥基丁酸酯的半衰期以及降低人為因素的干擾，我在小鼠飲水中給予β-羥基丁酸酯的前驅物1,3-丁二醇，之後再以鼻腔注射方式給予Alternaria alternata萃取物引發呼吸道過度反應，實驗結果也發現能夠有效地抑制真菌萃取物所引發的呼吸道發炎。接下來，我們透過給予小鼠生酮飼料來更真實模擬透過攝食來提升體內β-羥基丁酸酯濃度，並一樣以鼻腔注射方式給予Alternaria alternata萃取物引發過敏反應，結果發現生酮飼料能夠有效提升並維持血清中β-羥基丁酸酯濃度，也觀察到血清生理數值的變化，最重要的是能有效地降低真菌萃取物所引起第二型過敏反應。 基於以上小鼠疾病模式的結果，我們想進一步探討β-羥基丁酸酯對肺部ILC2細胞影響的機制，因此我們對小鼠肺部分選出的ILC2細胞給予β-羥基丁酸酯的處理，結果發現β-羥基丁酸酯並不會直接抑制經介白素-33處理的ILC2細胞分泌第二型細胞激素，而由於先前的研究指出β-羥基丁酸酯能降低肥大細胞的去顆粒作用(Mast cell degranulation)，因此我們給予經小鼠骨髓分化而來的細胞β-羥基丁酸酯的處理，結果發現能有效地抑制其產生第二型細胞激素以及介白素-2，並減少去顆粒化相關基因的表現。因此，我們推測β-羥基丁酸酯可能透過抑制肥大細胞產生的促發炎因子來進一步抑制ILC2分泌第二型細胞激素，實驗結果也證實細胞培養液中的第二型細胞素有明顯減少的趨勢。本篇研究證實，透過提升體內環境中β-羥基丁酸酯的濃度，能夠有效抑制由ILC2所引發的呼吸道發炎，並提供了攝取生酮飲食來提升體內β-羥基丁酸酯的濃度，或許能夠改善呼吸道過敏反應的方法，但β-羥基丁酸酯所介導的抑制效果並不直接作用於ILC2細胞上，而可能是透過抑制肥大細胞所產生的促發炎因子而造成的效果，但詳細的機制仍有待進一步的研究。

Beta-hydroxybutyrate (BHB) is one of the metabolites of fatty acids. In mammals, it is the most abundant ketone body and can serve as an alternative resource for adenosine triphosphate (ATP) production. In humans, the physiological concentration of BHB is typically in the low μM range. However, under fasting state or ketogenic diet (KD), the concentration of BHB can reach up 2 mM. It has been shown that BHB exerts protective function in neurological diseases in animal models. Also, previous studies have shown that elevation of serum BHB may alleviate hypersensitivity. However, the mechanism remains unclear. Thus, we would like to investigate the effect of BHB on the allergic immune response. Our data showed that giving BHB exogenously can inhibit IL-33-induced lung inflammation through suppressing Th2 cytokine production by ILC2s. Next, in order to give exogenous BHB through less manual intervention and solve short half-life of BHB, mice were administered with BHB precursor, 1,3-butanediol, through drinking water and treated with Alternaria alternata intranasally to induce airway hyperreactivity. The results indicated that elevation of serum BHB through drinking water can alleviate A. alternata-induced AHR and lung inflammation. In addition, we wondered whether elevation of BHB concentration through KD can alleviate Alternaria alternata-induced lung inflammation. Our results showed that administration of KD can elevate BHB concentration to 1 mM in the serum and alleviate airway hyperreactivity and inflammation. However, when we treated lung sorted ILC2s with BHB in vitro, the results showed that BHB did not inhibit cytokine production of ILC2s. Based on a study showed that BHB had suppressive effects on mast cell degranulation, so we wondered if BHB can also affect cytokine production of mast cells. Our data showed that a significant suppression on cytokine production of BM derived mast cells treated with BHB. Moreover, the gene expression of mast cell protease 1 (Mcpt1) and histidine decarboxylase (Hdc) were also decreased in mast cells treated with BHB. These data suggested that BHB had suppressive effects on mast cell activation. Finally, to investigate whether the suppressive effects of BHB on ILC2s in vivo are through interaction between mast cells and ILC2s, we co-cultured lung ILC2s with BM mast cells in the presence or absence of BHB. Our data showed that a significant reduction of Th2 cytokines, such as IL-5 and IL-13, from ILC2s was correlated with a great decreasing level of IL-2 and IL-9 from mast cells. This data indicated that BHB may inhibit Th2 cytokine production of ILC2s via decreasing IL-2 and IL-9 produced from mast cells. Overall, this study demonstrated that elevating the level of the ketone body, beta-hydroxybutyrate (BHB), in the circulatory system can alleviate ILC2-dependent airway inflammation and the suppressive effects of BHB may be through inhibiting mediators produced from mast cells to regulate effector functions of ILC2s.