腸病毒A71型 (Enterovirus A71，EV-A71)感染能夠在孩童中樞神經系統造成嚴重的疾病及併發症。一些高調節於血清或腦脊髓液的發炎或毒性因子，例如TNF-α, IL-1β, IL-6, IL-8, IL-10, IL-13和IFN-γ，與臨床疾病發展有高度相關，而且可更可因腦炎惡化。實驗室先前已指出在病毒感染的SF268星狀細胞中，減抑(knockdown)補體蛋白受器(globular complement 1q receptor，gC1qR)表現能提升TNF-α, IL-6, IL-10, 和 IL-13。反之，gC1qR 過度表達（overexpression）可抑制病毒產成，意味著gC1qR可扮演一個負向調控因子來調節病毒的複製與細胞激素的反應。為了更深入探討gC1qR是否在腸病毒A71型感染或病變扮演一個保護的角色，我們利用微量注射方式將h-gC1qR-knockdown 或-overexpression慢病毒載體 (lentiviral vector)分別注入受精卵，用以建立基因轉殖小鼠。然而只有h-gC1qR過度表達之轉殖鼠被建立成功，h-gC1qR減抑之轉殖鼠的失敗可能歸因於h-gC1qR之shRNA序列無法有效抑制小鼠內源性gC1qR(m-gC1qR)，且h-gC1qR與m-gC1qR大約只有82%的同源性。因此，過度表達h-gC1qR轉殖鼠(EV/gC1qR+)與野生型小鼠上(EV/WT)被用來感染或不感染一種經小鼠適性後的腸病毒A71株(MP4-M2)，並監測感染後出現之病理症狀是否與血清及周邊與中樞神經器官的病毒產量及細胞激素產成有相關聯。小鼠大約於感染後第五天開始出現與臨床相關較嚴重的病徵，包含後肢無力、癱瘓、甚至死亡。相較於EV/WT小鼠，EV/gC1qR+小鼠展現較佳存活率，同時有較高體重及低病徵指標分數。出現病徵的小鼠，其周邊器官(muscle和intestine)與脊隨病毒量均比大腦(不含腦幹)與腦幹都還要早出現峰值，意謂著病毒是由周邊器官或神經逆行軸突且經由脊隨傳遞到中樞神經系統。然而相較於EV/WT小鼠，只有在第五天時EV/gC1qR+小鼠的中樞神經系統包含大腦(不含腦幹)、腦幹及脊隨，其病毒產量降低了約10倍。同樣的於第五天相較於EV/WT小鼠，EV/gC1qR+小鼠血清中TNF-α, IL-6的表現量均顯著降低，意謂著h-gC1qR可能具有降低發炎反應的能力。另外在EV/gC1qR+小鼠上， IL-1β 和IL-13在大腦(不含腦幹)顯著上升； IL-6, IL-10和 IFN-β在腦幹顯著降低； IFN-β在脊隨顯著降低。同時，IL-1β 及IFN-γ上升及TNF-α和IL-6降低的趨勢也發生在EV/gC1qR+小鼠的中樞神經器官，顯示在EV/gC1qR+小鼠的中樞器官有緩減的發炎反應，並有上調的發炎小體及Type II適應性免疫反應。在EV/WT及EV/gC1qR+小鼠之周邊器官的比較下，沒有任何的臨床相關細胞激素有調節上的顯著差異。然而在EV/gC1qR+小鼠的周邊與中樞器官之細胞激素的表現量有著緩減的發炎與第一型干擾素的反應，並有上調發炎小體及第二型干擾素反應的趨勢。最後，在組織病理學中，可以發現h-gC1qR轉殖鼠呈現較少的組織破壞、細胞核外掉、及充血情形。綜合上述結果，我們認為h-gC1qR在周邊器官及中樞神經上，有減少病毒量及偏向於調節抗發炎與抗病毒細胞激素的角色。

EV-A71 infection causes severe complications in the central nervous system (CNS) in young children. Elevated inflammatory or toxic cytokines, such as TNF-α, IL-1β, IL-6, IL-8, IL-10, IL-13 and IFN-γ in serum and cerebrospinal fluids are highly correlated to clinical disease progression and can be promoted by brainstem encephalitis. Our lab has previously shown that knockdown of human-gC1qR (h-gC1qR) enhanced TNF-α, IL-6, IL-10, and IL-13 expression from infected glioblastoma SF268 cells. Overexpression of h-gC1qR in contrast to knockdown in SF268 cells reduced virus production, suggesting that gC1qR acts as a negative modulator for viral replication and/or cytokine responses. To investigate whether gC1qR has a role in protecting EV-A71 infection and/or pathogenic outcome, transgenic mice were established by microinjection of h-gC1qR-knokdown or -overexpression lentiviral vector into fertilized eggs. However, only h-gC1qR-overexpression but not h-gC1qR-knockdown mice were produced, presumably due to ineffectiveness of the shRNA with only 82% sequence homology to silence mouse-gC1qR (m-gC1qR). Consequently, h-gC1qR-overexpresson mice and wild-type mice (WT) were challenged with or without a mouse-adapted EV-A71 strain to monitor disease outcomes and to correlate viral titers as well as cytokines in serum and organs from peripheral and CNS organs. After challenge, severely-infected mice appeared some clinical-resembling syndromes around the 5th day of postinfection (dpi), including weakness, one or two hind-limb paralysis, or death. Infected h-gC1qR-overexpression mice (EV/gC1qR+) showed better survivor rate with higher bodyweights and less disease scores than infected WT mice (EV/WT). Viral titers in diseased mice were all peaked earlier in peripheral organs (muscle and intestine) and spinal cord than in brain and brain stem, suggesting retrograde axonal transmission from peripheral organs/nerves to CNS via the spinal cord. However, comparing to EV/WT mice, EV/gC1qR+ mice had 10-fold less viral titer only at 5 dpi in peripheral organs as well as in CNS including brain without brain stem (BWOB), brain stem, and spinal cord. Comparing with EV/WT mice at 5 dpi, EV/gC1qR+ mice had significantly lower levels of TNF-α and IL-6 in serum with implication of decreased proinflammation response, while with significant higher IL-1β and IL-13 in BWOB, significant lower IL-6, IL-10, and IFN-β in brain stem, as well as significant lower IFN-β in spinal cord. There were also indicative trends of upmodulated IL-1β and IFN-γ as well as downmodulated TNF-α and IL-6 in the CNS of EV/gC1qR+ mice, suggesting that EV/gC1qR+ mice had decreased proinflammation with increased inflammasome and adaptive immune responses in the CNS. None of these clinical-relevant cytokines in peripheral organs of EV/WT mice were significantly modulated in those of EV/gC1qR+ mice. However, EV/gC1qR+ mice had indicative trends of cytokines in decreasing proinflammation and type-I interferon responses and promoting inflammasomal and type-II interferon responses in the peripheral organs as in the CNS. Finally, histopathology examination revealed that EV/gC1qR+ mice had less tissue disruption, necrotizing myositis, and congestion than EV/WT mice. These findings together indicated that h-gC1qR has a role to protect mice from EV-A71-induced diseases by reducing viral loads and modulating a biased cytokine response toward anti-inflammation and antiviral activities in the peripheral and CNS organs.

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