Gr-1+CD11b+ 細胞特性鑑定於不同致病力之H1N1流感病毒感染小鼠

Characterization of Gr+CD11b+ cells in different pathogenic strains of H1N1 influenza virus infections

羅敏(Ming Lo)

指導教授 ： 蔡錦華

共同指導教授 ： 林素珍(Sue-Jane Lin)

國立臺灣大學/醫學院/微生物學研究所/碩士(2013年)

https://doi.org/10.6342/NTU.2013.10080

摘要

流感病毒感染是全球性重要的公衛議題。目前施行的預防性疫苗以及抗病毒藥物對於降低大規模流感病毒爆發的風險雖有一定成效，然而高致病力流感病毒感染所造成的重症病患亟需發展針對降低宿主過度免疫反應的治療策略來減少流感病毒造成的死亡。
細胞激素風暴在流感病毒致病機轉中扮演關鍵角色。本論文利用已建立之三株不同致病力H1N1 流感病毒小鼠感染模式，藉由比較致死性的A/PR8-1/34 (PR8)、中度致病性的A/Taiwan/126/09 以及致病性最低的A/Taiwan/141/02 感染小鼠肺臟的免疫反應，嘗試找出其中細胞激素或細胞吸引素的來源細胞；並探討不同株流感病毒感染中細胞激素風暴如何造成宿主不同嚴重程度的呼吸道發炎反應而導致對宿主致病力的差異。之前研究已利用蛋白質微陣列分析找到病毒感染小鼠肺泡支氣管沖洗液中特定細胞激素與細胞吸引素的表現量與病毒致病力的正向關連。本論文首先利用RT-PCR顯示病毒感染小鼠肺臟浸潤的白血球中會表現蛋白質微陣列分析中與病毒致病力呈正相關的細胞激素與細胞吸引素的mRNA。在高致病力的PR8感染小鼠體內，進一步利用流式細胞儀分析發現Gr1+CD11b+細胞佔肺臟內白血球最高的比例。型態上，PR8感染小鼠肺臟內的Gr1+CD11b+ 細胞絕大部分帶有大量明顯的質內空泡；且此群細胞高度表現細胞激素與細胞吸引素相關的基因顯示其高度活化的狀態。本研究發現在流感病毒感染小鼠中Gr1+CD11b+ 細胞是三種單核球吸引素：CCL2、CCL7 以及 CCL12的主要細胞來源。分子機制上，在第一型干擾素受器剔除小鼠感染流感病毒後肺臟內的Gr1+CD11b+ 細胞中CCL2、CCL7 以及 CCL12基因表現顯著被抑制的結果暗示第一型干擾素調控Gr1+CD11b+ 細胞產生單核球吸引素。肺臟中越多Gr1+CD11b+ 細胞累積，病毒感染小鼠血清中CCL2、CCL7 以及 CCL12的蛋白質濃度越高。推測因此較多骨髓球（myeloid cells）從骨髓中被吸引致肺臟累積。本研究揭示在流感病毒感染小鼠中，Gr1+CD11b+ 細胞是產生CCL2、CCL7 以及 CCL12的主要細胞來源，並提出Gr1+CD11b+ 細胞由骨髓移行至肺臟的可能途徑。以上資訊對未來發展高致病力流感病毒感染治療策略中，針對調節Gr1+CD11b+ 細胞在肺臟的累積有所助益。

關鍵字

Gr+CD11b+細胞 ； 流行性感冒病毒 ； 細胞激素風暴 ； 致病機轉

並列摘要

Influenza virus infection is a critical public health issue worldwide. While preventive vaccination and antiviral therapies are primary available approaches to lower risk of influenza virus outbreaks, as for high-mortality influenza pandemics, the need to identify supplementary mechanisms to alleviate overly-aggressive host response to influenza virus is urgent.
Cytokine storm plays a significant role in influenza virus infection. In this study, we try to define the cellular source and the mechanism how cytokine storm contributes to different pathological outcomes caused by different strains of influenza virus by using established model of three strains of H1N1 influenza viruses with distinct pathogenecities infections in mice: A/PR8-1/34 (PR8) as fatal strain, A/Taiwan/126/09 as moderately pathogenic strain and A/Taiwan/141/02 as mildly pathogenic strain. Previous results show the positive correlation between virus strains pathogenecities and the production level of specific cytokines and chemokines in protein array analysis of bronchoalveolar lavage fluid in virus-infected mice. In this thesis, we first demonstrate the mRNA expression of those cytokines and chemokines in pulmonary infiltrating leukocytes of virus-infected mice by RT-PCR. In PR8-infected mice, we further identify Gr1+CD11b+ cells as dominant infiltrating leukocytes by flow cytometry. Morphologically, most Gr1+CD11b+ cells in lung possess significant cytoplasmic vacuoles in PR8 infected mice. In addition, the upregulated gene expression profile of chemokines and cytokines in pulmonary infiltrating Gr1+CD11b+ cells in PR8 infected mice indicates its hyperactivated status. We found these cells primarily contribute to the production of monocyte chemotractants: CCL2, CCL7 and CCL12. The failure of the induction of those chemokines in Gr1+CD11b+ cells from virus-infected IFNAR KO mice hints out the possible regulation role of Type 1 IFN. While more above chemokines are produced by pulmonary infiltrating Gr1+CD11b+ cells, the serum concentration of above chemokines are elevated according to the amount of Gr1+CD11b+ cells in lung. As a result, it is suggested that more monocytes are recruited from bone marrow to lung. This study discovers Gr1+CD11b+ cells as a major cellular source of CCL2, CCL7 and CCL12 in influenza virus infection in mice and proposes their possible migration route from bone marrow to lung. Above information may help us develop potential therapeutic strategies by manipulating the accumulation of pulmonary infiltrating Gr1+CD11b+ myeloid cells in highly pathogenic influenza virus infections.

並列關鍵字

Gr+CD11b+ cells ； influenza virus ； cytokine storm ； pathogenesis

參考文獻

Aldridge, J. R., Jr., Moseley, C. E., Boltz, D. A., Negovetich, N. J., Reynolds, C., Franks, J., Brown, S. A., Doherty, P. C., Webster, R. G. & Thomas, P. G. (2009). TNF/iNOS-producing dendritic cells are the necessary evil of lethal influenza virus infection. Proc Natl Acad Sci U S A 106, 5306-5311.

Arankalle, V. A., Lole, K. S., Arya, R. P., Tripathy, A. S., Ramdasi, A. Y., Chadha, M. S., Sangle, S. A. & Kadam, D. B. (2010). Role of Host Immune Response and Viral Load in the Differential Outcome of Pandemic H1N1 (2009) Influenza Virus Infection in Indian Patients. Plos One 5.

Arimori, Y., Nakamura, R., Yamada, H., Shibata, K., Maeda, N., Kase, T. & Yoshikai, Y. (2013). Type I interferon limits influenza virus-induced acute lung injury by regulation of excessive inflammation in mice. Antiviral Res.

Baskin, C. R., Bielefeldt-Ohmann, H., Tumpey, T. M., Sabourin, P. J., Long, J. P., Garcia-Sastre, A., Tolnay, A. E., Albrecht, R., Pyles, J. A., Olson, P. H., Aicher, L. D., Rosenzweig, E. R., Murali-Krishna, K., Clark, E. A., Kotur, M. S., Fornek, J. L., Proll, S., Palermo, R. E., Sabourin, C. L. & Katze, M. G. (2009). Early and sustained innate immune response defines pathology and death in nonhuman primates infected by highly pathogenic influenza virus. Proc Natl Acad Sci U S A 106, 3455-3460.

Bender, A., Albert, M., Reddy, A., Feldman, M., Sauter, B., Kaplan, G., Hellman, W. & Bhardwaj, N. (1998). The distinctive features of influenza virus infection of dendritic cells. Immunobiology 198, 552-567.