探討過敏性氣喘的免疫及分子調控機制

Study on Immune and Molecular Regulation of Allergic Asthmatic Disease

黃心穎(Hsin-Ying Huang)

指導教授 ： 江伯倫

國立臺灣大學/醫學院/免疫學研究所/博士(2009年)

https://doi.org/10.6342/NTU.2009.00298

摘要

過敏性氣喘起因於無害過敏原所引起的異常第二型T細胞（Th2）反應，這類異常的Th2反應被認為與第一型（Th1）與第二型T細胞反應的不平衡有關。嗜酸性白血球聚集（eosinophilia）在過敏性氣喘致病機制中中扮演重要的角色，且其嚴重程度與細胞激素interleukin 5 (IL-5）和趨化素eotaxin 的增加有關。嗜酸性白血球（eosinophils）所釋出之物質會傷害呼吸道上皮細胞、引起黏液的分泌以及平滑肌收縮進而引起呼吸道過渡反應（airway hyperresponsiveness, AHR）。IL-5對於嗜酸性白血球的從骨髓中分化、從骨髓釋出至周邊血以及嗜酸性白血球在組織中的活化和存活皆有重要的影響，而eotaxin則為吸引嗜酸性白血球至發炎組織和促進其於組織中活化及存活主要的趨化素。目前氣喘的治療方式主要是利用單株抗體或是蛋白質抑制劑來調節過敏反應，然而相較於直接調控蛋白質的功能，透過調控mRNA可以更有效調節基因表現。而近年來研究發現，RNA干擾（RNA interference）可以專一並有效抑制基因表現。本研究欲設計IL-5和eotaxin的專一性小片段RNA（short hairpin RNA, shRNA）並利用慢病毒載體（lentivirual vectors）將shRNA送入氣喘模式小鼠中，探討IL-5和eotaxin的shRNA在呼吸道發炎中的角色。首先，利用體外系統確認shRNA可以有效抑制IL-5以及eotaxin表現；進一步將帶有IL5 shRNA（IL5SEC4）或eotaxin shRNA（eoSEC3.3）的表現基因組（shRNA expressing cassette, SEC）的lentivirus在以卵清蛋白（ovalbumin, OVA）刺激小鼠前，以氣管注射（intratracheal, i.t.）的方式送入以OVA誘導氣喘的小鼠中。結果顯示局部、單次單獨給予IL5SEC4或eoSEC3.3皆可有效緩減呼吸道發炎反應，包括減緩嗜酸性白血球聚集和AHR，並有效減少肺泡沖洗液中IL-5和eotaxin的含量。且若合併給予兩種shRNAs（IL5SEC4＋eoSEC3.3）亦可有效緩減氣喘模式小鼠的發炎反應，但相較於給予單一種shRNA之小鼠，合併兩種shRNAs並沒有更好的治療效果。儘管給予IL-5 或eotaxin shRNA可以有效抑制呼吸道發炎，但是對於血清中過敏原專一性免疫球蛋白E（immunoglobulin E, IgE）並無影響。
另外，Th1細胞及調節性T細胞（regulatory T cells, Tregs）被認為可以有效調控過敏原所引起之Th2反應；因此，在過敏氣喘的治療策略上，除了阻斷Th2反應外，誘導Th1細胞和Tregs是目前積極被研究作為氣喘治療的方向。研究指出修飾T細胞的抗原決定位（epitope peptide）之單一或數個胺基酸所產生之異體胜肽（altered peptide ligands, APLs）可能會大幅改變T細胞的功能，並指出APLs可以透過產生不同的細胞激素或是促使T細胞分化成不同型態來調節免疫反應。因此本研究欲利用OVA專一性的T細胞接受器（T cell receptor）基因轉質小鼠（DO11.10）之CD4 T細胞來研究APLs對於T細胞功能上的影響。本研究針對DO11.10 T細胞的主要epitope peptide加以修改，設計了六段單一胺基酸修飾的APLs（E333A，H331Q，H331F，H331R，H331E和 N335A），並以這些APLs刺激DO11.10 T細胞並測試T細胞的型態。其中五段APLs （E333A，H331Q，H331F，H331R和H331E）完全無法誘導T細胞增生、細胞激素分泌以及TCR內吞（internalization）。而N335A則可以在高濃度下刺激T細胞活化，由此結果推測N335A可能為弱增效劑（weak agonist）而其他五段APLs則為無效胜肽（null peptide）。此外，相較於WT刺激過之T細胞，N335A刺激過之T細胞在以WT peptide刺激下會分泌高量細胞激素IFN-γ以及較低的IL-4，推測N335A可能會誘導T細胞分化為Th1細胞。然而，六段APLs刺激過之T細胞完全沒有表現任何Treg相關之基因或功能。
本研究結果顯示局部給予IL-5或eotaxin shRNAs有潛力成為緩減呼吸道發炎的治療方式，並顯示可利用針對引起氣喘過敏反應之細胞激素或趨化素的shRNAs作為臨床治療方式可能性。而從體外試驗也找出可以誘導T細胞分化為Th1細胞之APL，接下來將把APL送入氣喘模式小鼠體內，以了解是否在體內APL是否可以達到改善過敏免疫的目的並了解是否可以透過誘導調節性免疫反應而有效地抑制呼吸道的發炎反應。

關鍵字

氣喘 ； RNA干擾 ； 介白質-5 ； 嗜伊紅趨化原 ； 異體胜肽

並列摘要

Asthma is caused by aberrant Th2 responses to harmless inhaled allergens underlaid a disturbance in the balance between Th1- and Th2-mediated immune responses. Eosinophilia plays the major role in the pathogenesis of asthma and correlates with the up-regulation of eotaxin, which, together with interleukin (IL)-5, is important for differentiation, chemo-attraction, degranulation, and survival of eosinophils in local tissue. Mediators released by eosinophils directly damage the epithelium cells and induce smooth muscles constriction and mucus secretion, subsequently result in airway hyperresponsiveness. Many therapeutic strategies for asthma are based on modulating the allergic responses through antibody or inhibitor. However, gene regulation in mRNA levels is more efficient than in protein levels. Recently, RNAi has been shown to be an efficacious in transcriptional or posttranscriptional silencing target genes. The present study aimed to investigate the role of eotaxin and IL-5 short hairpin RNAs (shRNAs) and their synergistic effect on airway inflammation in an ovalbumin (OVA)-induced murine model of asthma. Lentivirus-delivered shRNAs were used to suppress the expression of eotaxin and/or IL-5 in local tissue and we found that intra-tracheal administration of lentivirus containing eotaxin or IL-5 shRNAs expressing cassette (eoSEC3.3 or IL5SEC4) efficiently moderated the characteristics of asthma, including airway hyper-responsiveness, cellular infiltration of lung tissues, and eotaxin and IL-5 levels in bronchio-alveolar lavage fluid. concomitant administration of lentiviruses expressing IL-5 and eotaxin shRNAs (IL5SEC4 + eoSEC3.3) also moderated the symptoms of asthma in a mouse model. However, there was no effect on OVA-specific IgE level neither in single shRNA treated mice nor combination of eotxin and IL-5 shRNAs treated ones.
In addition, Th1 cells and regulatory T cells are thought to modulate the allergen-induced Th2 responses. Peptide alterations of T cell epitopes with single or few amino acid variations can have drastic effects on the outcome of this recognition. Altered peptide ligands (APLs) can act as modulators of immune responses through induction of different cytokines production profile or differentiation of different lineage of T cells. In this study, we would like to design APLs, which modified from OVA323-339 peptide, major epitope of DO11.10 TCR transgenic mice, and investigate whether APLs affect the function of allergen-specific T cells, such as differentiating toward Treg or Th1 and then we will investigate whether APLs could modulate the airway inflammation in OVA-induced murine of asthma mice, as an allergen-specific immunotherapy approach. We synthesized six APLs with a single amino acid substitution of the OVA323-339 (WT) and examined the phenotypes of OVA-specific T cells activated by these peptide analogues. In our results, five of six peptide analogues (E333A, H331Q, H331F, H331R, and H331E,) did not induce proliferation, TCR internalization, and cytokines production of DO11.10 T cells, being null peptides. Whereas, N335A induced activation of T cells in high concentration, likely being a weak agonist. Furthermore, N335A-primed T cells produced higher level of INF-γ and lower level of IL-4 compared with WT-primed T cells upon WT restimulation, indicating that N335A likely skewed T cells toward Th1 cells. However, T cells stimulated with six peptide analogues did not show any properties of Treg cells.
In our study, local delivery of lentiviruses expressing IL-5 and eotaxin shRNAs provides a potential tool in moderating airway inflammation and also has the potential for developing clinical therapy based on the application of shRNAs of chemokines and cytokines involved in T helper 2 cell inflammation and eosinophilia. In addition, we found that the OVA peptide analogous, N335A, could induce OVA-specific T cells to differentiate into Th1 cells and then we will deliver N335A into OVA-sensitized mice to investigate whether N335A could regulate the allergic responses and inhibit the airway inflammation in murine model of asthma.

並列關鍵字

asthma ； shRNA ； IL-5, eotaxin ； altered peptide ligands

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