黃麴毒素B1對豬免疫細胞及豬第二型環狀病毒細胞內複製的影響

Abstract

黃麴毒素B1(AFB1)是常見於亞熱帶及熱帶地區飼料原料穀物的黴菌毒素，豬隻可能因攝食到黴菌汙染的飼料而接觸AFB1，由於長期接觸低劑量AFB1會影響動物的免疫系統，因此AFB1可能成為豬隻產生豬環狀病毒相關症(PCVAD)的環境因子。本研究自無臨床症狀的豬第二型環狀病毒(PCV2)感染豬隻分離周邊血液單核細胞(PBMCs)，並將細胞進一步分化為單核球來源的樹突細胞(MoDCs)以做為抗原呈獻細胞(APCs)的研究模式，檢測豬免疫細胞於in vitro條件下與AFB1共同後培養，對其細胞功能及PCV2複製的影響。在對免疫細胞功能影響的結果顯示，10 μg/ml 高濃度AFB1處理72小時會減少PBMCs受到致裂原Con A刺激後之細胞存活率及細胞增殖能力；10 μg/ml高濃度AFB1處理48小時會使MoDCs變小、變圓，並失去樹突狀結構，並會降低MoDCs的抗原攝取與處理能力；10 μg/ml高濃度AFB1處理24小時後會增加MoDCs的細胞表面抗原CD40、CD83及CD86，以及免疫抑制性細胞激素IL-10的 mRNA表現量；0.001 μg/ml 低濃度AFB1則在處理後6小時造成MoDCs的CD40及CD83 mRNA表現量上升。在對PCV2複製的影響上，PBMCs受到72小時致裂原Con A的活化下，雖然10 μg/ml AFB1處理並不影響整個處理單位內細胞中的總病毒量，但卻可能增加單位細胞內所含的PCV2病毒量；而MoDCs經過10 μg/ml AFB1處理72小時後，也有處理單位內細胞量減少但細胞中總病毒量與對照組含量相近的現象。本研究結果顯示10 μg/ml 高濃度AFB1有機會增加MoDCs及Con A活化之PBMCs單位細胞內的PCV2病毒複製、傷害細胞媒介性免疫中的APCs功能及細胞增殖能力，並與PCV2同樣能刺激細胞的IL-10 mRNA表現而造成免疫抑制，故可能促使攝食到飼料被高量AFB1汙染的PCV2次臨床感染豬隻發展為PCVAD患豬。

Aflatoxin B1 (AFB1) is a mycotoxin commonly found in cereal grain and related products in subtropical and tropical regions, and pigs may expose to this toxin through contaminated feed. Chronic, low-dose exposure of AFB1 is known to affect the immunity, and may be a potential environmental factor in association with porcine circovirus type 2 (PCV2) in the induction of porcine circovirus-associated disease (PCVAD). In this study, peripheral blood mononuclear cells (PBMCs) were isolated from healthy subclinically PCV2-infected pigs and further differentiated to monocyte-derived dendritic cells (MoDCs) as an antigen-presenting cells (APCs) model to investigate the changes in immunocyte functions and PCV2 replication after in vitro AFB1 exposure. The PBMCs viability and Con A-induced proliferation were impaired after treated with AFB1 at 10 μg/ml for 72 hours. The cell morphology of MoDCs became smaller or rounded, contained shorter dendrites, and lost the antigen uptaking and processing abilities after exposure to 10 μg/ml AFB1 for 48 hours. The mRNA expression levels of surface markers CD40, CD83, and CD86 and regulatory cytokine IL-10 increased in MoDCs after treated with 10 μg/ml AFB1 for 24 hours. A significantly up-regulated CD40 and CD83 mRNA levels were observed in MoDCs after treated with 0.001μg/ml AFB1 for 6 hours. The cell-based PCV2 load appeared to increase in 10 μg/ml AFB1-treated Con A co-treated PBMCs, although no significant difference was seen in the total copy number of PCV2 genome; similar increase in cell-based PCV2 load might also occurr in 10 μg/ml AFB1-treated MoDCs. These results indicate that a relatively higher concentration of AFB1 such as 10 μg/ml may have the potential to increase PCV2 replication in PBMCs and MoDCs, impair the APCs cell function and cell proliferation in cell-mediated immunity, and cause immunosuppression through enhancement of IL-10 mRNA expression. Thus, it is suggested that AFB1 may potentiate PCVAD development in subclinically PCV2 infected pigs through dietary exposure to AFB1.