Characterization of the lipid kinase activity of the p110β mutant.

<p>(A) HEK 293T cells were transfected with p85 and wild type or E633K myc-p110β. Anti-myc immunoprecipitates were analyzed by western blotting and for lipid kinase activity. (B) Anti-myc immunoprecipitates from cells transfected as above were incubated for 2 hours with pY-peptide and assayed for lipid kinase activity. (C) Anti-myc immunoprecipitates from cells transfected as above were incubated with lipid vesicles/Gβ₁γ₂ subunits for 10 minutes and assayed for lipid kinase activity. (D) Sequence alignment of p110α, p110β, p110γ and p110δ focusing on the acidic patch containing the E633 p110β residue, highlighted in red. (E) Specific activity of wild-type and D626K p110α co-expressed with p85 in HEK 293T cells and assayed as above. All data are mean ± SEM of triplicate determination from three separate experiments.</p

Effect of p110β mutant on transformation and chemotaxis.

<p>(A) NIH 3T3 cells stably expressing wild type or E633K p110β were plated in soft agar and colonies were counted after 3 weeks. Colony counts are normalized to the number of colonies produced by cells expressing p110β alone. (B) Equal number of NIH 3T3 cells stably expressing wild type or E633K p110β were plated and left to grow to confluence for 10 days. Foci were counted and normalized to cells expressing wild-type p110β. (C) NIH 3T3 cells stably expressing wild type or E633K p110β were starved overnight and plated either in 0% or 10% NCS in transwell chambers, and incubated with media containing 0% or 10% NCS in the lower chamber and upper chambers as indicated. Data are mean ± SEM of triplicate samples from two experiments.</p

Akt signaling, proliferation and survival of cells expressing mutant p110β.

<p>(A) Expression level of wild-type or E633K myc-p110β in stably-transfected cells. (B) Cells stably expressing wild type or E633K p10β were incubated overnight in 10%, 0.5% or 0% NCS media. Whole cell lysates were analyzed by western blotting with anti-pT308 Akt, anti-pT389 S6K, and anti-β-actin antibodies. (C-E) Cells stably expressing wild-type or E633K p110β were plated in 96-well plates, incubated for 24 and 48 hours in (C) 10% NCS medium, (D) 0.5% NCS medium, or (E) 0% NCS medium, and assayed using the MTT assay. (F) Cells stably expressing wild type or E633K p110β were incubated for 24 hours in 10%, 0.5%, or 0% NCS medium. Cell viability was assayed by Trypan blue staining. Dead cells are displayed as percent of total number of cells. Data are mean ± SEM of triplicate samples from two separate experiments.</p

Role of kinase activity on the increased proliferation and migration by the p110β mutant.

<p>(A) Cells stably expressing wild-type or E633K p110β were plated in 96-well plates, incubated for 24 and 48 hours in 10% NCS, with or without 200 nM TGX-221, and assayed using the MTT assay. (B) NIH 3T3 cells stably expressing wild type or E633K p110β were starved overnight and plated either in 0% or 10% NCS in transwell chambers, and incubated with media containing 0% or 10% NCS in the lower chamber and upper chambers as indicated, with or without 200 nM TGX-221 in both chambers as indicated. Data are mean ± SEM of at least duplicate samples from two separate experiments.</p

p110γ/δ Double-Deficiency Induces Eosinophilia and IgE Production but Protects from OVA-Induced Airway Inflammation

<div><p>The catalytical isoforms p110γ and p110δ of phosphatidylinositide 3-kinase γ (PI3Kγ) and PI3Kδ play an important role in the pathogenesis of asthma. Two key elements in allergic asthma are increased levels of eosinophils and IgE. Dual pharmacological inhibition of p110γ and p110δ reduces asthma-associated eosinophilic lung infiltration and ameliorates disease symptoms, whereas the absence of enzymatic activity in p110γ^KOδ^D910A mice increases IgE and basal eosinophil counts. This suggests that long-term inhibition of p110γ and p110δ might exacerbate asthma. Here, we analysed mice genetically deficient for both catalytical subunits (p110γ/δ^-/-) and determined basal IgE and eosinophil levels and the immune response to ovalbumin-induced asthma. Serum concentrations of IgE, IL-5 and eosinophil numbers were significantly increased in p110γ/δ^-/- mice compared to single knock-out and wildtype mice. However, p110γ/δ^-/- mice were protected against OVA-induced infiltration of eosinophils, neutrophils, T and B cells into lung tissue and bronchoalveolar space. Moreover, p110γ/δ^-/- mice, but not single knock-out mice, showed a reduced bronchial hyperresponsiveness. We conclude that increased levels of eosinophils and IgE in p110γ/δ^-/- mice do not abolish the protective effect of p110γ/δ-deficiency against OVA-induced allergic airway inflammation.</p></div

Additional file 2: Figure S2. of Deficiency of PI3-Kinase catalytic isoforms p110γ and p110δ in mice enhances the IL-17/G-CSF axis and induces neutrophilia

Expression of CXCR4 and CXCL12/SFD-1α in WT, p110γ−/−, p110δ−/−, and p110γ/δ−/− mice. a To measure CXCL12/SDF-1α concentrations in the BM, tibias were flushed with 500 μl PBS, cells were pelleted and supernatants were subsequently subjected to ELISA. Bars represent means + SD of n = 9–10 mice per group. b To determine CXCR4 expression leukocyte suspensions were labeled with fluorescent antibodies and analyzed by flow cytometry. Neutrophils were gated as singlet, live CD3ε − CD19− CD11b+ Siglec-F− Ly6G+ cells and were analyzed for the expression of CXCR4 (CD184). Shown are GMFI of CD184-APC of gated neutrophils in BM (left), blood (middle) and spleen (right). Bars represent means + SD of n = 5–8 mice per group. c To determine CXCR2 expression leukocyte suspensions were labeled with fluorescent antibodies and analyzed by flow cytometry. Neutrophils were gated as singlet, live CD3ε − CD19− CD11b+ Siglec-F− Ly6G+ cells and were analyzed for the expression of CXCR2 (CD182). Shown are GMFI of CD182-APC of gated neutrophils in BM (left), blood (middle) and spleen (right). Bars represent means + SD of n = 7–8 mice per group. (JPEG 248 kb

Lung tissue infiltration by eosinophils, neutrophils, T and B cells is only reduced in OVA-treated p110δ^-/- and p110γ/δ^-/- mice.

<p>To determine OVA-induced infiltration of immune cell populations into the lung tissue, leukocytes were prepared from lungs after BAL and exsanguination of PBS-treated and OVA-treated KO and corresponding WT mice. Cell populations were analysed by flow cytometry. Cell counts were normalised as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159310#pone.0159310.g002" target="_blank">Fig 2</a>. (<b>A</b>) Eosinophils (eos) in lung tissue from p110γ^-/- and WT mice (left), p110δ^-/- and WT mice (middle), and p110γ/δ^-/- and WT mice (right). (<b>B</b>) Neutrophils (neutros) in lung tissue from p110γ^-/- and WT mice (left), p110δ^-/- and WT mice (middle), and p110γ/δ^-/- and WT mice (right). (<b>C</b>) T cells in lung tissue from p110γ^-/- and WT mice (left), p110δ^-/- and WT mice (middle), and p110γ/δ^-/- and WT mice (right). (<b>D</b>) B cells in lung tissue from p110γ^-/- and WT mice (left), p110δ^-/- and WT mice (middle), and p110γ/δ^-/- and WT mice (right). Data (n = 3–6) are presented as means + SD. Data were analysed by One-way ANOVA followed by Bonferroni’s comparison tests for selected pairs of columns. ⁺⁺⁺ P < 0.001, ⁺⁺ P < 0.01, ⁺ P < 0.05. ⁺ indicate differences between WT PBS and WT OVA groups. ***P < 0.001, **P < 0.01, *P < 0.05. Asterisks indicate differences between OVA-treated groups.</p

Bronchoalveolar infiltration of eosinophils, neutrophils, T and B cells is reduced in OVA-treated p110γ^-/-, p110δ^-/-, and p110γ/δ^-/- mice.

<p>To determine the number of eosinophils, neutrophils, T and B cells in the BALF from OVA-treated and PBS-treated KO and corresponding WT mice, cells were collected, and analysed by flow cytometry. Cell counts were normalised as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159310#pone.0159310.g002" target="_blank">Fig 2</a>. (<b>A</b>) Eosinophils (eos) in BALF from p110γ^-/- and WT mice (left), from p110δ^-/- and WT mice (middle), and from p110γ/δ^-/- and WT mice (right). (<b>B</b>) Neutrophils (neutros) in BALF from p110γ^-/- and WT mice (left), p110δ^-/- and WT mice (middle), and p110γ/δ^-/- and WT mice (right). (<b>C</b>) T cells in BALF from p110γ^-/- and WT mice (left), p110δ^-/- and WT mice (middle), and p110γ/δ^-/- and WT mice (right). (<b>D</b>) B cells in BALF from p110γ^-/- and WT mice (left), p110δ^-/- and WT mice (middle), and p110γ/δ^-/- and WT mice (right). Data (n = 3–6) are presented as means + SD. Data were analysed by One-way ANOVA followed by Bonferroni’s comparison tests for selected pairs of columns. ⁺⁺⁺ P < 0.001, ⁺⁺ P < 0.01, ⁺ P < 0.05. ⁺ indicate differences between WT PBS and WT OVA groups. ***P < 0.001, **P < 0.01, *P < 0.05. Asterisks indicate differences between OVA-treated groups.</p

Bronchial hyperresponsiveness and goblet cell metaplasia are reduced in OVA-treated p110γ/δ^-/- mice.

<p>To determine bronchial hyperresponsiveness, lung function analysis was performed using the IPL and changes in airway resistance were measured following systemic application of rising doses of methacholine (MCh). Some values had to be excluded, e.g. when lungs were damaged during the experiments. Changes in airway resistance in (<b>A</b>) PBS-treated (n = 3–10) and (<b>B</b>) OVA-treated (n = 5–7) KO and WT mouse groups. All three WT groups were analyzed and pooled for a clearer graphical presentation. Data in (<b>B</b>) were analysed by Two-way ANOVA followed by Bonferroni’s comparison tests *P < 0.05. (<b>C, D</b>) Mucus production in PBS-treated and in OVA-treated KO and WT mice. To measure mucus production, lungs were collected after IPL and cut into 6 μm thick slices. Sections were stained for carbohydrates using the periodic acid-Schiff (PAS) reaction and counter stained with H&E. Representative lung tissue sections from WT, p110γ^-/-, p110δ^-/-, and p110γ/δ^-/- mice after (<b>C</b>) PBS-treatment and (<b>D</b>) OVA-treatment. Magnification 100x, inserts 630x. (<b>E</b>) PAS⁺ cells (pink) per basement membrane in mm. Bars express means + SD; Data (n = 3–6 mice) were analysed by One-way ANOVA followed by Tukey’s Multiple Comparison Test; ***P < 0.001.</p

Nucleotide-independent extraction of Rac1^Ic from the liposomes by GDI1.

<p>(<b>A</b>) GST-GDI1 pull-down of Rac1^Ic but not of Rac1<i>^Ec</i>. Input is the total mixture of beads and proteins, and output is the pull-down (PD). (<b>B</b>) Liposome binding of Rac1^Ic but not of Rac1<i>^Ec</i>. In the liposome sedimentation assay, Rac1^Ic efficiently binds to liposomes in the absence of GDI1 and independent of whether it was loaded with GDP or GppNHp, a non-hydrolysable GTP analog. Rac1<i>^Ec</i> failed to bind to liposomes under the same conditions. (<b>C</b>) Preferential binding Rac1^Ic to GDI1 than to liposomes. GDI1 binds to both GDP-bound and GppNHp-bound Rac1^Ic proteins and prevents their association with the liposomes. (<b>D</b>, <b>E</b>) GDI1 efficiently extracted GDP-bound Rac1^Ic from the liposomes and to a lower extend also Rac1^Ic-GppNHp. Same amount of GDP-bound and GppNHp-bound forms of Rac1^Ic associated with the liposomes were prepared before incubation with 5-fold molar excess of GDI1 and sedimentation at 20,000x<i>g</i> (<b>D</b>). Using increasing molar excess of GDI1 (2-, 5-, 10-, 15- and 20-fold) showed that higher concentrations of GDI1 are required to extract Rac1^Ic-GppNHp from the liposomes to supernatants in comparison to Rac1^Ic-GDP (<b>E</b>). CBB, coomassie brilliant blue; <i>Ec</i>, <i>E. coli</i>; Ic, insect cells; P, liposome pellet; S, supernatant.</p