探討免疫基因多型性與免疫細胞表現之相關性和利用體外免疫突觸系統篩檢有效疫苗佐劑之研究

Discovering SNPs in Immunity-Related Genes to Enhance Disease-Resistance in Pigs and Characterizing the Porcine Immunological Synapse as an In Vitro Fast Screening Platform for Potential Vaccine and Adjuvant Candidates

陳映安(Ann Ying-An Chen)

指導教授 ： 莊秀琪

國立屏東科技大學/獸醫學院/獸醫學系所/博士(2021年)

https://doi.org/10.6346/NPUST202200005

摘要

提高抗病性和對病菌之耐受性於經濟動物配種時為一個重要選拔目標。免疫系統中的抗原表現會受到基因多型性的影響，當受到病原菌感染時會因基因多型性的影響使抗原變異進而影響免疫調節與免疫優勢之抗原。單核苷酸多型性（SNP）是指多個核酸位點上會產生變異而改變基因表達，SNP的產生會影響個體的免疫調節以及對疾病的敏感度。免疫突觸 （immunological synapse, IS）是在感染性損傷或接種疫苗後於抗原呈現細胞（APC）和淋巴細胞之間形成的一種特殊結構。在細胞間進行交互作用時，細胞藉由訊息傳導路徑進行交流並觸發信號傳遞訊息，最終產生持久的免疫力。本次研究分兩部分，將調查免疫相關基因中SNP 位點的分佈及於不同的豬隻品種中 : 純種豬（台灣黑豬、杜洛克、藍瑞斯和約克夏）和雜交豬（藍瑞斯-約克夏）其體內免疫細胞表面標誌的關聯性。本研究中以豬隻IS模式將 APC 與 T 細胞亞群共培養，用以初步篩選豬繁殖與呼吸綜合徵病毒 （PRRSV） 疫苗於接種後是否為潛在之有效疫苗和佐劑。免疫相關基因有 39 個 SNP，包括細胞激素（IFN-α、IFN-γ、TNF-α）、趨化因子（GM-CSF、MCP-1） 和類鐸受體(TLR3、TLR4、TLR7、TLR8 和 TLR9），實驗中從具有CD4、CD8、CD80/86、MHCI 和 MHCII表面標記的細胞進行分析。實驗中，將十二頭藍瑞斯-約克夏混種之無特定病原 (SPF) 豬分成 3 組，對照組和接種自行開發之PRRSV-1 或 PRRSV-2 疫苗兩組。 28 天後，採自實驗豬隻之周邊血液分選成四種T 細胞亞群（Tn 細胞：CD4+CD25-、NK 細胞：CD4-CD25+、Tregs、CD4+CD25+ 和陰性對照組細胞：CD4-CD25-）各別同步與肺泡巨噬細胞共培養 12 小時。周邊血液單核球與肺泡巨噬細胞共培養的結果可分析 Toll-like receptor mRNA 之基因表現和細胞激素之分泌量。其中有 28 個 SNP 在品種之間存在顯著差異，尤其是純種豬中的藍瑞斯豬和台灣黑豬之間。例如，於SNP1中測得 IFN-α -235A/G 在台灣黑豬和藍瑞斯豬中的頻率分別為 11.11% 和 96.15%。另外也有18個SNP與細胞表面標誌的表達有顯著相關，包括CD4、CD8、CD80/86和MHCII，例如SNP33 中測出TLR-8 543C/C 中 CD4+ 的百分比為39.27% 明顯高於 A/C 之24.34 %，p<0.05。本次研究能夠觀察到免疫突觸的三個階段結合後之形態特徵。兩組接種疫苗之組別，其 Toll-like receptor （TLR） 3、4、7、8 和 9 均顯著上升。相對於對照組，接種PRRSV-2 疫苗組於共培養實驗中所產生之 IL-10分泌量較低。最後，各種T 細胞也表現出不同的 TLR 表達模式。總結本篇研究顯示，台灣黑豬具有獨特的基因型分佈，而藍瑞斯豬和約克夏豬之基因型分佈則較為相似。因此，探討不同物種間與免疫調節關聯之SNP找出其基因獨特性，並可進一步運用於豬隻之育種選拔。豬隻IS模式系統未來可應用於初步篩選開發中疫苗/佐劑或找出有效之抗原決定位。本實驗之最終目標，是使本篩選平台可能有助於在疫苗開發的初步階段時減少實驗動物的使用。

關鍵字

基因多型性 ； 細胞表面標記 ； 免疫突觸 ； T 細胞亞群 ； 豬

並列摘要

Enhancing resistance and tolerance to pathogens remains an important selection objective in the production of livestock animals. Host-pathogen interactions underlie the selection of antigenic variants through genetic polymorphisms of immune responses; impacting immunoregulation and immunodominant antigens. Single nucleotide polymorphisms (SNPs) vary gene expression on the transcriptional level, influencing an individual’s immune regulation and susceptibility to diseases. The immunological synapse (IS) is specialized cell to cell junction that forms between lymphocytes and antigen presenting cells (APC) following an infectious insult or vaccination. Upon interaction, these cells communicate and trigger signal transduction pathways which ultimately establishes long-lasting immunity. This study consisted of two parts, we investigated the distribution of SNP sites in immune-related genes and their correlations to cell surface markers of immune cells within, purebred (Taiwan black, Duroc, Landrace and Yorkshire) and crossbred (Landrace-Yorkshire) pigs. For the first time, we characterized the porcine immunological synapse by co-culturing APC with T cell subsets to screen for potential effective vaccine and adjuvant candidates following vaccination against Porcine Reproductive and Respiratory Syndrome virus (PRRSV).Thirty nine SNPs of immune-related genes, including cytokines, chemokines and toll-like receptors (IFN-α, IFN-γ, TNF-α, GM-CSF, MCP-1, TLR3, TLR4, TLR7, TLR8, and TLR9) were selected and the percentages of positive cells with cell surface markers of CD4, CD8, CD80/86, MHCI, and MHCII were analyzed. Here, twelve Landrace-Yorkshire specific pathogen-free (SPF) pigs were separated into 3 groups, control and two groups vaccinated with either PRRSV-1 or PRRSV-2. After 28 days, four purified T cell subsets from peripheral blood (Tn cells; CD4+CD25-, NK cells; CD4-CD25+, Tregs; CD4+CD25+, and Mixed cells; CD4-CD25-) were co-cultured for 12 hours with pulmonary alveolar macrophages. Toll-like receptor mRNA expression patterns and cytokine levels were analyzed. There were 28 SNPs that were significantly different among breeds, particularly between Landrace and Taiwan black. For instance, the frequency of SNP1 IFN-α -235A/G in Taiwan black and Landrace were 11.11% and 96.15%, respectively. In addition, 18 SNPs significantly correlated with the expressions of cell surface markers, including CD4, CD8, CD80/86, and MHCII. The percentage CD4+ (39.27%) in SNP33 TLR-8 543C/C was significantly higher than those in A/C (24.34%), at p<0.05. Also, three stages of the IS were characterised by morphological features following conjugation. Results showed a significant upregulated in Toll-like receptors (TLR) 3,4,7,8 and 9 in both vaccinated groups. Further, a significantly lower level of IL-10 was produced in co-cultures with PRRSV-2 compared to controls. Finally, T cells also exerted different TLR expression patterns. Together, our findings showed that Taiwan black pigs had a unique genotype distribution, whereas Landrace and Yorkshire had a more similar genotype distribution. Thus, an understanding of the genetic uniqueness of each breed could help identify functionally important SNPs in immunoregulation. These findings demonstrate that the porcine immunological synapse can be characterized in vitro to screen for microenvironment factors, with future applications in vaccine/adjuvant candidate or effective antigen epitopes. Ultimately, this method could potentially aid in reducing the use of experimental animals during the preliminary stages of vaccine development.

並列關鍵字

single nucleotide polymorphisms ； cell surface markers ； immunological synapse ； T cell subsets ； pig

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