探討幽門螺旋桿菌熱休克蛋白 60 對人類周邊血液單核球抑制增生活性之位置

Abstract

因幽門螺旋桿菌所造成慢性感染已被公認會導致消化道方面的 疾病，像是慢性胃炎、消化性潰瘍、黏膜相關淋巴組織淋巴癌 (MALT lymphoma)，甚至有可能發展成胃癌。最近，根據報導由幽門螺旋桿 菌所分泌的熱休克蛋白 60 與胃部的發炎具有相關性。並且幽門螺旋 桿 菌 的 熱 休 克 蛋 白 60 已 被 證 實 可 誘 導 許 多 前 發 炎 激 素(pro-inflammatory cytokines)，例如 TNF-α、IL-8、IL-6 以及 IFN-γ。 有趣的是，有些文獻指出不同物種的熱休克蛋白 60 與免疫調控有相 關性。舉例來說，日本血吸蟲所分泌的熱休克蛋白 60 可誘導調控 T 細胞(Treg)的產生並達到抑制發炎的效果。而我們實驗室的先前研究 也顯示幽門螺旋桿菌的熱休克蛋白 60 可降低人類周邊血液單核球細 胞 (human peripheral blood mononuclear cells, PBMCs)之增生活性，且 抑制前發炎激素的分泌，並是經由誘導細胞週期的停滯進而影響 PBMCs 的增生。這些發現指出幽門螺旋桿菌的熱休克蛋白 60 可能具 有兩種功能，分別是誘導發炎與免疫調控。根據文獻顯示幽門螺旋桿菌的熱休克蛋白 60 主要是經由 TLR-2 的路徑來造成發炎。另一方 面，幽門螺旋桿菌的熱休克蛋白 60 其胺基酸位置 300-435 也被證實 具有可誘導單核球細胞分泌 IL-8 的能力。然而，幽門螺旋桿菌的熱 休克蛋白 60 是由哪一個區域的胺基酸位置來誘發免疫調控仍然還是 未知。因此，我的方針為探討幽門螺旋桿菌的熱休克蛋白 60 的哪一 個位置具有免疫調控的能力。 為了達到這個目的，所以我們建構完整幽門螺旋桿菌的熱休克蛋 白 60 (胺基酸序列：1-547) 及五個不同的幽門螺旋桿菌熱休克蛋白 60 片段 (胺基酸序列：1-200、101-200、1-250、200-300 及 300-547)。 由於 Treg 細胞可經由 IL-10 或 TGF-β 這兩個細胞激素來抑制 CD4 + T 細胞的增生，所以我們藉由監控這兩個免疫抑制的細胞激素 (IL-10 及 TGF-β)來找出哪一個片段可誘發免疫調控。研究結果顯示，重組 的熱休克蛋白 60 (胺基酸序列：101-200 及 300-547)可誘導 IL-10 的分 泌。並經由可專一性辨識重組熱休克蛋白片段 60 (胺基酸序列： 101-200)的單株抗體進一步證實可負調控免疫抑制。這些結果顯示幽 門螺旋桿菌的熱休克蛋白 60 (胺基酸序列：101-200)可能與免疫抑制 有相關性。

Chronic infection with Helicobacter pylori (H. pylori) is recognized as the cause of upper gastrointestinal disorder, such as chronic active gastritis, peptic ulcer diseases, mucosa-associated lymphoid tissue (MALT) lymphoma and is an important risk factor for the development of gastric cancer. Recently, it was also reported that heat shock protein 60 of H. pylori (HpHsp60) are associated with gastric inflammation. Hsp60 of H. pylori has been found that it can induce immune cells to express the strong pro-inflammatory cytokines, such as TNF-α, IL-8, IL-6, and IFN-γ. Interestingly, some literatures indicated that Hsp60 derived from different species seem to be related to the regulation of immune responses. For example, the Hsp60 of S. japonicum can generate regulatory T cell (Treg) and suppress immunity. Our lab’s previous studies have shown that H. pylori Hsp60 can decrease human peripheral blood mononuclear cells (PBMCs) proliferation rate, inhibit cytokine secretion, and induce cell cycle arrest. These findings indicate that HpHsp60 may have two functions, induction of the inflammation and immune suppression. The mechanisms of HpHsp60-induced inflammation in which TLR-2 pathway is the major signaling pathway have been confirmed. On the other hand, the amino acid domains 300-435 of H. pylori Hsp60 is associated with induction of IL-8 in monocytic cell has been explored. What domain of the HpHsp60-induced immune suppression is still remains unknown. Thus, the aim in this study is to investigate which domains of HpHsp60 function for immune suppression. For the purpose of this study, we construct whole Hsp60 (amino acid sequence: 1-547) and five partial domains (sequence: a. a. 1-200, 101-200, 1-250, 200-300, 300-547). Since Treg cells inhibit proliferation of CD4+ T cells either via IL-10 or TGF-β production, we demonstrate which domains of HpHsp60 function for immune suppression by monitoring the two cytokines (IL-10 and TGF-β) expression. The preliminary data showed rHsp60s (sequence: a. a. 101-200 and 300-547) induced IL-10 secretion. The monoclonal Abs against rHsp60 (sequence: a. a. 101-200) to prove that rHsp60 (sequence: a. a. 101-200) could down-regulate the immune responses. These results propose that the sequence from 101 to 200 of H. pylori Hsp60 may be closely associated with immune suppression.