Importance of methodology in the evaluation of renal mononuclear phagocytes and analysis of a model of experimental nephritis with Shp1 conditional knockout mice
Importance of methodology in the evaluation of renal mononuclear phagocytes and analysis of a model of experimental nephritis with Shp1 conditional knockout mice
复制标题
方法学在评估肾单核吞噬细胞和Shp1条件敲除小鼠实验性肾炎模型分析中的重要性
DOI：
10.1016/j.bbrep.2020.100741
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发表时间：
2020
影响因子：
2.7
通讯作者：
Hiromura Keiju
中科院分区：
文献类型：
--
作者：
Watanabe Mitsuharu;Kaneko Yoriaki;Ohishi Yuko;Kinoshita Masato;Sakairi Toru;Ikeuchi Hidekazu;Maeshima Akito;Saito Yasuyuki;Ohnishi Hiroshi;Nojima Yoshihisa;Matozaki Takashi;Hiromura Keiju
组织驻留单核吞噬细胞（Mophs），包括单核细胞，巨噬细胞和树突状细胞（DCs），在生理和病理条件下发挥重要作用。这些细胞在肾脏中的存在已经知道了几十年，目前正在进行肾Mophs（rMophs）的研究。由于没有统一的程序已确定分离rMophs，流式细胞术分析的rMophs的结果一直不一致的研究。因此，我们首先评估了使用胶原消化的rMophs的制备方法。胶原酶消化后rMophs的产量大大增加。特别是F4/80 high rMophs，其对DC的特异性标志物CD 11 c呈阳性，显著增加。此外，由于在流式细胞术分析中中性粒细胞有时混合在rMophs中，我们建立了用于消除中性粒细胞的门控策略。为了确定rMophs对自身免疫性肾炎发展的贡献，我们分析了应用于Shp 1条件性基因敲除小鼠（Shp 1 CKO）的自身免疫性肾炎实验模型。通过将携带floxed Shp 1等位基因的小鼠品系与在CD 11 c启动子控制下表达Cre重组酶的小鼠杂交产生该敲除菌株。因此，Shp 1 CKO在表达CD 11 c的细胞中特异性缺乏Shp 1。结果表明，Shp 1 CKO对实验性肾小球肾炎易感，F4/80高表达的Shp 1 CKO细胞明显增多。总之，我们的制备方法的胶原酶消化和中性粒细胞的门控策略是必要的rMophs的分析，和Shp 1抑制自身免疫性肾炎的发展，通过控制rMophs。
Tissue resident mononuclear phagocytes (Mophs), comprising monocytes, macrophages, and dendritic cells (DCs), play important roles under physiological and pathological conditions. The presence of these cells in the kidney has been known for decades, and studies of renal Mophs (rMophs) are currently underway. Since no unified procedure has been identified to isolate rMophs, results of flow cytometric analysis of rMophs have been inconsistent among studies. We therefore first evaluated a preparative method for rMophs using collagenous digestion. The yield of rMophs greatly increased after the collagenase digestion. In particular, F4/80high rMophs, which were positive for CD11c, a specific marker of DCs, dramatically increased. In addition, since neutrophils are sometimes mixed among rMophs in the analysis of flow cytometry, we established a gating strategy for eliminating neutrophils. To determine the contribution of rMophs to the development of autoimmune nephritis, we analyzed an experimental model of autoimmune nephritis that was applied to Shp1 conditional knockout mice (Shp1 CKO). This knockout strain is generated by crossing a mouse line carrying floxed Shp1 allele to mice expressing Cre recombinase under the control of the CD11c promoter. Shp1 CKO therefore specifically lack Shp1 in cells expressing CD11c. As a result, Shp1 CKO were susceptible to that experimental glomerulonephritis and F4/80high rMophs of Shp1 CKO increased dramatically. In conclusion, our preparative methods for collagenase digestion and gating strategy for neutrophils are necessary for the analysis of rMophs, and Shp1 suppresses the development of autoimmune nephritis through the control of rMophs.