丙酮酸激?缺乏導致鐵含量過剩影響脾臟巨噬細胞對真菌的反應

Abstract

鐵在宿主與病菌間的交互關係中扮演兩種角色:一方面可以調控宿主的免疫反應，另一方面為病菌養分的提供者。本篇研究主要利用組織胞漿菌的感染模式探討丙酮酸激酶缺乏的小鼠免疫細胞的功能。在糖酵解循環中，丙酮酸激酶是一種常見的酵素並且缺乏此酵素會導致慢性非溶血性貧血以及肝臟和脾臟有鐵沉積的情形。至於丙銅酸激酶缺乏是如何影響免疫細胞的功能這個問題目前還未被討論。在這邊我使用中研院所提供的基因突變小鼠，這是經由ENU所導致pklr基因點突變形成丙銅酸激酶缺乏的小鼠P354，藉由此方式去了解丙銅酸激酶缺乏在感染的模式中是如何影響免疫反應。 本篇研究發現經由流式細胞儀分析得到P354小鼠脾臟細胞中TER119 紅血球前驅物占了很高的比例，但B淋巴球和T淋巴球占的比例卻比正常小鼠低了很多。然而P354小鼠T淋巴球在胸腺和淋巴結所占的比例是和正常小鼠沒有差異性的。此篇研究也證實P354小鼠的脾臟和肝臟有明顯的鐵沉積情形並且發現在感染組織胞漿菌後的不同天數，P354小鼠真菌數量在肝臟和脾臟而非肺臟都高於正常小鼠。更進一步地，P354小鼠在感染組織胞漿菌之後脾臟所產生TNF-alpha、IL-6、IFN-gamma、IL-4、IL-10、IL-17和T細胞所產生的IFN-gamma 量都比起正常小鼠低很多。除此之外，有趣地是我還發現雖然P354小鼠腹腔巨噬細胞的功能和正常小鼠沒有差異，但脾臟巨噬細胞在吞噬組織胞漿菌的功能上和產生TNF-alpha有明顯的缺失現象。以上實驗結果顯示，P354小鼠無法有效清除脾臟和肝臟中的組織胞漿菌而這兩個器官也剛好有鐵沉積的情形。脾臟清除能力下降其原因為細胞激素產量較低以及巨噬細胞的功能上有缺陷。雖然P354突變小鼠腹腔巨噬細胞的功能和正常小鼠沒有差異，但若外加全轉鐵蛋白給正常小鼠的腹腔巨噬細胞就會降低TNF-alpha產生的量。即使無法排除丙酮酸激酶缺乏的小鼠脾臟巨噬細胞吞入太多紅血球而造成免疫功能上的缺陷但我的研究結果可以得知是因為脾臟和肝臟鐵沉積而導致巨噬細胞功能上有缺陷因此直接或間接的影響清除細胞內病原的能力。

Iron is a critical element in host-pathogen interaction. It is both a modifier of cellular immune function and an essential nutrient for microbes. Here, I investigated immune cell functions in pyruvate kinase deficient mice following infection by Histoplasma capsulatum (HC). Pyruvate kinase (PK) deficiency is the most common enzyme abnormality of glycolysis causing chronic non-spherocytic haemolytic anemia and accumulation of iron in the liver and spleen. Whether pyruvate kinase deficiency affects immune cell functions has never been addressed. The availability of ENU-mutagenized mice P354 with point-mutated pklr gene presents an opportunity to study how pyruvate kinase deficiency affects immune response to infection. In this study, cytometric analysis of the splenic cell populations revealed elevated numbers of TER119-positive erythroid precursor cells and lower percentages of T cells and B cells in P354 mutant mice compared to wild-type mice. However, the T cell populations in the thymus and lymph nodes of P354 mutant mice were comparable to that in wild-type mice. I demonstrated in this study that there was iron accumulation in the spleen and liver of P354 mutant mice and found that fungal burdens in the spleen and liver but not that in the lung of P354 mutant mice were higher than in wild-type mice at different time points after Histoplasma infection. Furthermore, the levels of cytokines (TNF-α, IL-6, IFN-γ, IL-4, IL-10, and IL-17) and the numbers of IFN-γ-producing T cells were lower in P354 mutant mice than in wild-type mice. Interestingly, while peritoneal macrophages from P354 mutant mice were functionally comparable to wild-type mice, splenic macrophages obtained from P354 mutant mice were less competent in phagocytosis of Histoplasma and production of TNF-α. These results showed that mice with pyruvate kinase deficiency were inefficient to clear Histoplasma from the spleen and liver, coinciding with increased iron contents in these organs. Inefficiency to rid the spleen of fungus also correlated with lower cytokine production and impaired macrophage function. While the function of peritoneal macrophages from P354 mutant mice was comparable to that of wild-type mice, the addition of holo-transferrin to peritoneal macrophages from wild-type mice impaired their ability to produce TNF-α. Although the possibility of overcrowding effect has not been excluded, the results of my study suggest that iron overload as observed in the spleen and liver of mice with pyruvate kinase deficiency impairs the function of macrophages, thereby directly or indirectly, affects their ability to rid an intracellular pathogen.