Department of Pharmacology and Experimental Therapy, Institute of Experimental and Clinical Pharmacology and Toxicology and ICePhA mouse clinic, University of Tübingen, D-72074, Tübingen, Germany.
Institute of Biochemistry and Molecular Biology II, University of Düsseldorf, D-40225, Düsseldorf, Germany.
Cell Commun Signal. 2017 Jul 19;15(1):28. doi: 10.1186/s12964-017-0185-y.
Phosphoinositide 3-kinase γ (PI3Kγ) and PI3Kδ are second messenger-generating enzymes with key roles in proliferation, differentiation, survival, and function of leukocytes. Deficiency of the catalytic subunits p110γ and p110δ of PI3Kγ and PI3Kδ in p110γ/δ mice leads to defective B- and T-cell homeostasis. Here we examined the role of p110γ and p110δ in the homeostasis of neutrophils by analyzing p110γ, p110δ and p110γ/δ mice.
Neutrophils and T cells in leukocyte suspensions from the bone marrow (BM), blood, spleen and lung were analyzed by flow cytometry. Serum concentrations of IL-17, of the neutrophilic growth factor G-CSF, and of the neutrophil mobilizing CXC chemokines CXCL1/KC and CXCL2/MIP-2 were measured by Bio-Plex assay. Production of G-CSF and CXCL1/KC by IL-17-stimulated primary lung tissue cells were determined by ELISA, whereas IL-17-dependent signaling in lung tissue cells was analyzed by measuring Akt phosphorylation using immunoblot.
We found that in contrast to single knock-out mice, p110γ/δ mice exhibited significantly elevated neutrophil counts in blood, spleen, and lung. Increased granulocytic differentiation stages in the bone marrow of p110γ/δ mice were paralleled by increased serum concentrations of G-CSF, CXCL1/KC, and CXCL2/MIP-2. As IL-17 induces neutrophilia via the induction of G-CSF and CXC chemokines, we measured IL-17 and IL-17-producing T cells. IL-17 serum concentrations and frequencies of IL-17 splenic T cells were significantly increased in p110γ/δ mice. Moreover, IFN-γ, IL-4, and IL-5 T cell subsets were drastically increased in p110γ/δ mice, suggesting that IL-17 T cells were up-regulated in the context of a general percentage increase of other cytokine producing T cell subsets.
We found that p110γ/δ deficiency in mice induces complex immunological changes, which might in concert contribute to neutrophilia. These findings emphasize a crucial but indirect role of both p110γ and p110δ in the regulation of neutrophil homeostasis.
磷酸肌醇 3-激酶 γ（PI3Kγ）和 PI3Kδ 是产生第二信使的酶，在白细胞的增殖、分化、存活和功能中起关键作用。PI3Kγ 和 PI3Kδ 的催化亚基 p110γ 和 p110δ 在 p110γ/δ 小鼠中的缺陷导致 B 细胞和 T 细胞的稳态受损。在这里，我们通过分析 p110γ、p110δ 和 p110γ/δ 小鼠，研究了 p110γ 和 p110δ 在中性粒细胞稳态中的作用。
通过流式细胞术分析骨髓（BM）、血液、脾脏和肺部白细胞悬浮液中的中性粒细胞和 T 细胞。通过 Bio-Plex 测定法测定血清中白细胞介素 17（IL-17）、中性粒细胞生长因子 G-CSF 以及中性粒细胞趋化因子 CXCL1/KC 和 CXCL2/MIP-2 的浓度。通过 ELISA 测定 IL-17 刺激的原代肺组织细胞产生的 G-CSF 和 CXCL1/KC，通过使用免疫印迹测定法分析肺组织细胞中 IL-17 依赖性信号转导来分析 Akt 磷酸化。
与单敲除小鼠相比，我们发现 p110γ/δ 小鼠血液、脾脏和肺部的中性粒细胞计数明显升高。p110γ/δ 小鼠骨髓中粒细胞分化阶段增加，同时血清 G-CSF、CXCL1/KC 和 CXCL2/MIP-2 浓度增加。由于白细胞介素 17 通过诱导 G-CSF 和 CXC 趋化因子诱导中性粒细胞增多，我们测量了白细胞介素 17 和产生白细胞介素 17 的 T 细胞。p110γ/δ 小鼠的血清白细胞介素 17 浓度和脾脏白细胞介素 17 T 细胞频率均显著升高。此外，IFN-γ、IL-4 和 IL-5 T 细胞亚群在 p110γ/δ 小鼠中急剧增加，表明在其他细胞因子产生 T 细胞亚群的百分比普遍增加的情况下，白细胞介素 17 T 细胞被上调。
我们发现 p110γ/δ 在小鼠中的缺陷诱导了复杂的免疫变化，这可能共同导致中性粒细胞增多。这些发现强调了 p110γ 和 p110δ 在调节中性粒细胞稳态中具有关键但间接的作用。
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