The role of STAT3 inhibitors in Type I IFN-mediated signaling and antiviral responses

先天性免疫系統(innate immunity)中，第一型干擾素在幫助宿主對抗病毒及細菌感染扮演很重要的角色。第一型干擾素會活化訊息傳導與轉錄子(STAT)蛋白，包括 STAT1、STAT2 以及 STAT3。相較於 STAT1 和 STAT2 主要是促進第一型干擾素引起的抗病毒反應，STAT3在其中則是扮演了一個負調控的角色。之前的研究報告指出，STAT3 基因剔除的鼠胚胎纖維母細胞(MEF)以及骨髓衍生的巨噬細胞(BMM)可表現較強的第一型干擾素反應，所以如果能抑制 STAT3 功能將可以提升干擾素引起的抗病毒作用。因此，我們篩選不同的 STAT3 抑制劑並研究其在調控第一型干擾素的訊息及功能上所扮演的角色。我們發現在第一型干擾素的刺激之下，WP1066 能有效的提高正常的小鼠胚胎纖維母細胞(MEFs)及小鼠骨髓衍生的巨噬細胞(BMMs)的抗病毒相關基因的表現。而這個現象在 STAT3 基因剔除細胞上是觀察不到的，這證實了抑制劑所造成的現象是因為作用在 STAT3 上。我們更進一步利用腦心肌炎病毒(EMCV)感染小鼠胚胎纖維母細胞(MEFs)，試驗 WP1066 對於第一型干擾素所引起抗病毒反應的影響。隨著 WP1066 藥物濃度的增加，被感染細胞內的 EMCV 基因會逐漸下降，這暗示了細胞的抗病毒反應有效被提升。此外，我們也發現在 STAT3 基因轉殖的細胞中，WP1066同樣可以消弭 STAT3 在第一型干擾素調控訊息中的負調控機制。另一方面，我們去探討了 WP1066 的作用機制，根據實驗結果發現，這個 STAT3 抑制劑並不會影響到第一型干擾素引發的 STAT1 和 STAT2 的磷酸化以及入核的能力。總而言之，綜合以上的結果，我們推論出 WP1066 可以專一抑制住STAT3的活性，進而提升細胞的抗病毒反應。這些結果也提供了一個治療病毒感染的模式，利用STAT3抑制劑去強化第一型干擾素的抗病毒反應。Type I interferons (IFNs) plays a key role in innate immunity to protect host from viral and bacterial infections. Signal transducer and activator of transcription (STAT) proteins, such as STAT1, STAT2, and STAT3, are activated by IFN-α/β. Unlike the complex of STAT1 and STAT2, which promotes type I IFN-mediated antiviral response, STAT3 negatively regulates type I IFN-mediated pathway. In previous study, STAT3 knockout mouse fibroblast (MEFs) and primary bone-marrow-derived macrophages (BMMs) showed enhanced IFN functions. We hypothesize that targeting STAT3 function would enhance IFN-mediated antiviral response. Therefore, we screened different STAT3 inhibitors and investigated their roles in IFN-mediated signaling and functions. Among them, WP1066 was shown to induce higher expression of antivirus-associated genes in both WT MEFs and BMMs in response to IFN-α4 stimulation. Interestingly, the phenomenon was abolished in the absence of STAT3, suggesting that the effect of the inhibitor was STAT3-dependent. The effect of WP1066 in IFN-α4-mediated antiviral response was further examined by infecting MEFs with encephalomyocarditis virus (EMCV). Enhanced antiviral response was revealed by reduced expression of EMCV-specific gene in infected cells in a dose-dependent manner. In addition, the suppressive effect of FL- and N-terminal domain- (NTD) STAT3 in STAT3KO MEFs in response to type I IFN was reversed by WP1066 treatment. We further addressed the mechanism of WP1066 activity and found that the inhibitor did not affect phosphorylation and nuclear translation of STAT1 and STAT2. Taken together, these results suggest that WP1066 may enhance antiviral function of cells by targeting STAT3 function. This study provides a therapeutic approach for virus infection by targeting STAT3 to enhance type I IFN-induced antiviral response