Abstract
BackgroundPhosphoinositide 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.MethodsNeutrophils 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.ResultsWe 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.ConclusionsWe 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.
Highlights
Phosphoinositide 3-kinase γ (PI3Kγ) and PI3Kδ are second messenger-generating enzymes with key roles in proliferation, differentiation, survival, and function of leukocytes
To analyze the intracellular expression of cytokines in T cells, splenocytes were incubated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA), 1 μg/mL ionomycin, and 5 μg/mL Brefeldin A in cell culture medium (RPMI supplemented with 10% FCS, 2 mM glutamine, 0.05 mM βmercaptoethanol, and 100 U/mL each of penicillin and streptomycin; all PAA, Pasching, Austria) at 37 °C for 3.5 h
We found that frequencies and total numbers of neutrophils in the bone marrow (BM) of p110γ−/−, p110δ−/−, and p110γ/δ−/− mice were similar to WT mice (Fig. 1b)
Summary
Phosphoinositide 3-kinase γ (PI3Kγ) and PI3Kδ are second messenger-generating enzymes with key roles in proliferation, differentiation, survival, and function of leukocytes. Neutrophil granulocytes are the most abundant circulating phagocytes and serve as first line of innate immune defense in microbial infections. They produce a wide range of antimicrobial effectors including reactive oxygen species like superoxide radicals and a variety of antibacterial proteins. The homeostasis of neutrophils needs to be tightly regulated, as acquired or idiopathic neutropenia predisposes to infections, whereas activation and accumulation of increased numbers of neutrophils can promote tissue damage in inflammatory disorders, such as severe asthma, COPD or rheumatoid arthritis [1, 2]. The most important growth factor for neutrophils is G-CSF, which directly regulates their proliferation and differentiation [3, 4]. Cellular sources of IL-17 are IL-17-producing CD4+ T helper cells (Th17) [6], some CD8+ T cells [7], and various innate immune cell types [8]
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