具影響干擾素γ 分泌的食材對OVA致敏BALB/c小鼠免疫反應的影響

Abstract

干擾素γ (interferon-gamma, IFNγ) 為第一型T輔助細胞 (Type I T helper cell, Th1) 所分泌的細胞激素，能夠抑制Th2細胞的分化、Th2細胞激素的分泌、B細胞生成IgE抗體的能力，可透過調節Th1/Th2平衡而降低過敏反應。此外，遭受感染或外來抗原侵入時，IFNγ 能活化巨噬細胞等吞噬細胞清除外來物，被活化的巨噬細胞會分泌多種發炎介質，促進發炎反應。過敏性氣喘為最常見的第一型過度免疫反應疾病 (Type I hypersensitivity)，體內有較正常者高的IgE抗體含量並傾向Th2反應，吸入過敏原時，呼吸道會有嚴重的發炎反應與eosinophil聚集現象，造成呼吸道過度反應 (airway hyperresponsiveness, AHR)，為過敏性氣喘主要症狀。已有許多研究指出，能夠透過飲食調節免疫反應，減緩過敏性氣喘的症狀，故本研究欲探討在過敏性氣喘小鼠模式下，經由飲食介入具有影響IFNγ 分泌的食材，對過敏性氣喘小鼠體內的過敏反應與呼吸道發炎反應有何影響？ 本研究首先利用初代脾臟細胞與ASEA與SMPC共同培養24小時，結果in vitro下分泌IFNγ 的能力，苜蓿芽乙酸乙酯萃取物 (Alfalfa sprouts EA extracts, ASEA) 為顯著抑制，SMPC (Stolle milk protein concentrate, SMPC) 為顯著促進，故以此兩種食材進行動物實驗。 過敏性氣喘小鼠模式依照食材介入時間分成兩部分，致敏後晚期餵食15天 (晚期餵食) 與致敏過程中開始餵食50天 (早期餵食)。BALB/c小鼠於8週齡時，以OVA伴隨佐劑Al(OH)3隔週腹腔注射致敏共三次。晚期餵食模式於第三次注射OVA後14天，以血清OVA特異性IgE含量分組；早期餵食模式於第一次注射OVA後5天以血清總IgE含量分組，共分為control組，低劑量ASEA 10與高劑量ASEA 20、低劑量SMPC 2與高劑量SMPC 4、固醇類藥物prednisolone正對照組 (Pred.)、PBS組，共七組。餵食期間已5%霧化之OVA給予呼吸道刺激，誘發過敏性呼吸道發炎反應，15週齡時以methacholine刺激呼吸道測定AHR，16週齡時犧牲並測定各種過敏免疫指標。 晚期餵食高低劑量ASEA皆可顯著降低AHR與肺氣管沖洗液 (BALF) 中IL-13、IFNγ 含量，並抑制脾臟細胞OVA刺激下IFNγ、IL-4分泌，ASEA 20組還具有降低血清中OVA特異性IgE與IgG1抗體含量的趨勢。早期餵食高低劑量ASEA依然可顯著抑制AHR，並可減少呼吸道嗜伊紅性白血球聚集，ASEA 10可顯著降低脾臟細胞OVA特異性IL-5分泌，ASEA 20則可顯著降低脾臟細胞IL-5與IL-13分泌量與OVA特異性IgG1抗體含量。晚期餵食SMPC可顯著抑制脾臟細胞OVA刺激下IFNγ、IL-4分泌量與血清中OVA特異性IgE和IgG1抗體含量，改善AHR並降低BALF中IFNγ 的含量，SMPC 4組可顯著降低BALF中IL-13與嗜伊紅性白血球細胞數，能更顯著降低AHR。早期餵食SMPC僅能降低BALF中IFNγ 的含量，但無法因此降低AHR，此外，可能因早期餵食SMPC造成免疫耐受性，使各種過敏指標皆無顯著影響。 由本實驗結果得知，給予氣喘小鼠餵食ASEA並不會促進Th2反應，而餵食SMPC並無法增加此疾病模式小鼠脾臟細胞IFNγ 的分泌量。晚期餵食模式下，兩種食材都可降低呼吸道發炎與抑制過敏反應，但以SMPC的效果較顯著，早期餵食模式下以ASEA降過敏反應的效果較佳。

IFNγ is secreted by Th1 cell and has been shown to inhibit the differentiation of Th2 cells, suppress the Th2 cytokine production, and decrease the ability of IgE production of B cell. IFNγ can also decrease allergic response by shifting Th1/Th2 balance toward Th1 immune response. During the infection, the phagocytes, such as macrophages, are activated by IFNγ to eliminate the pathogen. The activated macrophages can also release various kinds of inflammatory mediators to cause local inflammation. Allergic asthma is a common disease of type I hypersensitivity. Patients with allergic asthma are characterized with elevated IgE contents in serum which then act on Th2-prone immune responses. After exposing to the allergen, severe inflammation and eosinophilia will take place in airway, leading to airway hyperresponsiveness (AHR). Several studies have reported that dietary components have the potential of immunomodulation which can relieve the symptoms of allergic asthma. In this study, we used mouse model of allergic asthma to determine the effects of dietary components on IFNγ secretion in immune responses. First, primary splenocytes from BALB/c mice were treated with alfalfa sprout ethyl ester extracts (ASEA) or Stolle milk protein concentrate (SMPC) for 24 hours. ASEA could significantly decrease, while SMPC could significantly increase the IFNγ production by ConA-stimulated BALB/c splenocytes. Subsequently, mouse model of allergic asthma was used to determine the effects of tube-fed ASEA or SMPC on allergic responses in OVA-sensitized mice. Two parts of experiments were conducted according to the different feeding periods. In the first part, late-feeding, mice were grouped by OVA-specific IgE titer in serum after 3-times of OVA-sensitization, then tube-fed with ASEA or SMPC for 15 days. In the second part, early-feeding, mice were grouped by total IgE concentration in serum after one OVA-injection, followed by 50 days of tube-feeding. Mice were divided into 7 groups: control, ASEA 10, ASEA 20, SMPC 2, SMPC 4, prednisolone (Pred.) as positive control, and PBS as blank control. In both parts, female BALB/c mice were sensitized with OVA in Al(OH)3 adjuvant intraperitoneally at the age of 10, 12, and 14 week-old. During the feeding period, 5% OVA was inhaled by OVA-sensitized mice for 3 times to induce allergic airway inflammation. 24 hours after aerosol OVA challenge, the AHR of OVA-sensitized mice were measured at the age of 15 week-old. Mice were then sacrificed at the age of 16 week-old and BALF was collected 24 hours after aerosol OVA challenge. Late-feeding of ASEA, mice had decreased AHR, lowered IL-13 and IFNγ contents in BALF, and had lower IFNγ and IL-4 productions in splenocytes after OVA stimulation ex vivo. Mice fed with ASEA 20 also tended to decrease the titer of OVA-specific IgE and IgG1 in serum. Early-feeding of ASEA, mice had lower AHR and eosinophil number in BALF than control group. ASEA 10 could decrease the IL-5 production of OVA-stimulated splenocytes ex vivo. ASEA 20 could decrease not only IL-5, but also IL-13 production of OVA-stimulated splenocytes and OVA-specific IgG1 in serum. Late-feeding of SMPC, mice had significantly lowered IFNγ and IL-4 production of OVA-stimulated splenocytes ex vivo, lowered titers of OVA-specific IgE and IgG1 in serum, decreased AHR, and reduced levels of IFNγ in BALF. SMPC 4 could further decrease the contents of IL-13 and number of eosinophil in BALF, and consequently resulted in a much lowered AHR. On the other hand, early-feeding of SMPC, mice could only decrease the content of IFNγ in BALF without reducing AHR or any indicators of allergic asthma. This result may result from the immune tolerance induced by early-feeding of SMPC. In this study, we have showed that late-feeding of ASEA and SMPC have similar effects of reducing allergic response in OVA-sensitized mice, but SMPC is more effective. However, ASEA is a much better dietary component to down-regulate allergic response than SMPC in the early-feeding model.