B 細胞誘導調節性T 細胞對先天免疫細胞的調控機制研究

Abstract

巨噬細胞的過度活化與許多發炎疾病息息相關，像是痛風，糖尿病，乾癬症。因此若能並且給於適當的控制巨噬細胞的活化將會是治療發炎疾病重要的方法之一。近年來，研究結果發現，B 細胞誘導的調節性T 細胞，稱為Treg-of-B 細胞，具有表現出與調節性T 細胞相似的蛋白，並且具有調節後天免疫的能力，像是抑制T細胞的增生、第一型輔助性T細胞以及第十七型輔助性T 細胞的免疫反應。然而，關於Treg-of-B cells 是否調控先天免疫反應尚未探討。本篇研究的目的為探討Treg-of-B是否具有調控巨噬細胞的功能。 在研究結果中，我們發現在體外實驗 Treg-of-B 細胞，而非tTreg 或是effector T 細胞，方會抑制 LPS/ATP 刺激巨噬細胞引起的發炎體活化, 包括抑制IL-1 分泌, caspase-1 活化, 以及 NLRP3 的產生，並且是透過Treg-of-B 細胞與巨噬細胞之間的細胞接觸達到抑制的功; 我們更進一步發現Treg-of-B 細胞 藉由抑制NF-B 的活化來達到抑制巨噬細胞NLRP3 發炎體的活化。 由於之前的研究指出, M2 巨噬細胞NLRP3 發炎體相關的蛋白以及mRNA 表現會受到抑制，因此我們更進一步探討Treg-of-B 是否會影響巨噬細胞的極化。實驗結果發現，當巨噬細胞與Treg-of-B共同培養時，即使受到LPS/IFN- 的刺激，Treg-of-B細胞仍然會誘發巨噬細胞表現典型的M2 相關標記，像是Arg-1 、IL-10、Pdcd1lg2、MGL-1、IL-4、YM1/2、及CD206; 進一步探討機制的結果顯示，Treg-of-B 會藉由與巨噬細胞的細胞接觸，誘導巨噬細胞STAT6 的磷酸化,並且，實驗結果顯示在STAT6 缺陷的巨噬細胞，與Treg-of-B共同培養時，不能誘導巨噬細胞表現M2 相關標記。以上的結果證明Treg-of-B 細胞會藉由活化STAT6 的表現，進而促進M2 巨噬細胞的極化。 此外，我們建立小鼠皮下氣囊模式，探討尿酸結晶誘導NLRP3活化的發炎反應, 結果顯示Treg-of-B 細胞移植到小鼠體內能有效地減少發炎激素IL-1、 CXCL1、、及CCL2的產生，以及嗜中性球與單核球的浸潤。在痛風的動物模式中，我們觀察到, 浸潤囊腔的組織中Treg-of-B 細胞與巨噬細胞有細胞接觸的現象。因此，當小鼠受到尿酸結晶刺激時，Treg-of-B 細胞會移動到囊腔的組織，進一步抑制巨噬細胞NLRP3 發炎體的活化，減緩痛風疾病的發生。 另外，我們將Treg-of-B 誘導的M2 巨噬細胞以尾靜脈的方式給予小鼠，能夠有效的緩解imiquimod 誘導的小鼠乾癬症的病症，像是減緩小鼠皮膚的紅斑以及脫屑的現象。除此之外，給予Treg-of-B 誘導的M2 巨噬細胞能有效的抑制皮膚組織的IL-17F表現，以及抑制鄰近淋巴結細胞在體外刺激時分泌IL-22、IL-17、IFN-; 因此Treg-of-B cell 誘導的 M2 巨噬細胞, 會藉由減少局部以及系統性的發炎反應，達到減緩乾癬症。 綜合以上的實驗結果顯示，脾臟的B細胞所誘導的調節性T細胞能藉由抑制NLRP3 發炎體的活化, 以及誘導出M2 巨噬細胞, 來達到調控先天免疫的功能。此研究也提供未來使用Treg-of-B細胞治療發炎性或其他自體免疫疾病的潛力。

The excess activation of macrophage is associated with several inflammatory diseases, such as gouty arthritis, diabetes, and psoriasis. Hence, the proper regulation of the macrophage activation will be an important therapeutic strategy for inflammatory diseases. Recently, we identified a population of Foxp3- regulatory T cells that were induced by B cells, named Treg-of-B cells, which exert regulatory functions in adaptive immunity, including inhibition of Th1 and Th17-mediated immune responses. However, whether Treg-of-B cells regulate innate immunity remains unclear. This study aimed to investigate whether Treg-of-B cells could regulate macrophage function and differentiation. We showed that Treg-of-B cells, but not thymus-derived Treg or effector T cells, inhibited inflammasome-mediated IL-1 secretion, caspase-1 activation, and NLRP3 production by LPS/ATP stimulation in a cell contact-dependent manner. In addition, Treg-of-B cells inhibited monosodium urate-induced NLRP3 inflammasome activation in vitro via NF-B signaling. Previous study indicated that the induction of NLRP3-associated protein and mRNA expression is inhibited in M2 macrophages. Next, we further explored whether Treg-of-B cells could promote alternatively activated macrophage (M2 macrophages) polarization. The macrophages co-cultured with Treg-of-B cells upregulated typical M2-associated molecules, including Arg1, Il10, Pdcd1lg2, Mgl-1, Il4, Ym1/2 and Cd206 under M1 polarization condition. Further investigation of the molecular mechanism revealed that Treg-of-B cells promoted M2 macrophage polarization via STAT6 activation in a cell contact-dependent manner. Adoptive transfer of Treg-of-B cells ameliorated gouty inflammation in a mouse air pouch model through reducing neutrophil and leukocyte influx. Moreover, the mice intravenous receiving Treg-of-B cells had significantly lower levels of IL-1, CXCL1, and CCL2 compared to MSU-treated control mice. We also found that infiltrated Treg-of-B contacted with local macrophages, but not neutrophils in the tissue of air pouch. Therefore, Treg-of-B cells immediately migrated to air pouch and inhibited NLRP3 inflammasome activation, resulting in the amelioration of gouty arthritis upon MSU stimulation. Adoptive transfer of Treg-of-B-induced M2 macrophages in imiquimod-induced psoriasis mice alleviated skin inflammation such as reducing the erythema and scaling. Moreover, IL-17F mRNA expression in skin lesions was significantly decreased in Treg-of-B-induced-M2 macrophages treatment group. In addition, IL-22 and IFN-γ, and IL-17 production by immune cells in the draining lymph nodes was significantly reduced in the Treg-of-B-induced M2 groups. Therefore, Treg-of-B cell-induced M2 macrophages ameliorated manifestations of psoriasis by reducing local and systemic inflammation. Overall, our results demonstrated that Treg-of-B cells exerted regulatory effects on innate immunity by suppressing NLRP3 inflammasome activation and inducing alternatively activated macrophages, providing a cell-based therapeutic strategy for inflammatory diseases and autoimmunity diseases.