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

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

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

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

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

Groups of mice and their respective treatment following the injection schedule of Fig 1A.

<p>Groups of mice and their respective treatment following the injection schedule of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154001#pone.0154001.g001" target="_blank">Fig 1A</a>.</p

Inflammatory cells in BALF and lung tissue.

<p>(A) Mice were treated according to the injection schedule for the HDM-induced asthma model. (B) BALF was centrifuged and cells were analyzed via differential cell count. Differences remained non-significant. Data are presented as mean ± SEM; n = 3. (C) Representative micrographs of BALF cellspin preparations are shown (scale 100 μm, magnification x200). (D) Levels of neutrophils and eosinophils in lung tissue were measured via flow cytometry. Differences remained non-significant. Data are presented as mean ± SEM; n = 3.</p

mRNA-Mediated Gene Supplementation of Toll-Like Receptors as Treatment Strategy for Asthma <i>In Vivo</i>

<div><p>Asthma is the most common chronic disease in childhood. Although several therapeutic options are currently available to control the symptoms, many drugs have significant side effects and asthma remains an incurable disease. Microbial exposure in early life reduces the risk of asthma and several studies have suggested protective effects of Toll-like receptor (TLR) activation. We showed previously that modified mRNA provides a safe and efficient therapeutic tool for <i>in vivo</i> gene supplementation. Since current asthma drugs do not take patient specific immune and TLR backgrounds into consideration, treatment with tailored mRNA could be an attractive approach to account for the patient’s individual asthma phenotype. Therefore, we investigated the effect of a preventative treatment with combinations of <i>Tlr1</i>, <i>Tlr2</i> and <i>Tlr6</i> mRNA in a House Dust Mite-induced mouse model of asthma. We used chemically modified mRNA which is–in contrast to conventional viral vectors–non-integrating and highly efficient in gene transfer. In our study, we found that treatment with either <i>Tlr1/2</i> mRNA or <i>Tlr2/6</i> mRNA, but not <i>Tlr2</i> mRNA alone, resulted in better lung function as well as reduced airway inflammation <i>in vivo</i>. The present results point to a potentially protective effect of TLR heterodimers in asthma pathogenesis.</p></div

Tissue reaction and lung function after <i>Tlr</i> mRNA treatment.

<p>(A) Tissue inflammation and goblet cell metaplasia were analyzed on H&E- and PAS-stained lung sections. Representative micrographs are shown (original magnification of H&E sections: x200, scale 100 μm, PAS sections: x400). (B) PAS stained lung sections were analyzed to quantify PAS⁺ cells. Data are represented as individual mice, horizontal lines state means; n = 9. (C) Airway resistance was measured in response to rising concentrations of methacholine (MCh) using the isolated, perfused and ventilated lung (IPL). Statistical analysis was separately performed for each MCh concentration, * and # are PBS vs. the respective group, § is <i>Tlr1/2</i> vs. <i>Tlr2</i>. *<i>P</i> ≤ 0.05, **<i>P</i> ≤ 0.01, and ***<i>P</i> ≤ 0.001. Statistical results for each concentration can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154001#pone.0154001.s001" target="_blank">S1 Table</a>. (D) Data at 10 μM MCh are presented as Box and Whiskers blot. Medians are shown as +. Tukey’s Multiple Comparison Test **<i>P</i> < 0.01. Data are represented as means; data at 10 μM MCh are additionally stated as means ± SEM; n = 6 (<i>Tlr2</i> n = 5, <i>Tlr2/6</i> n = 4). Tabular values at 10 μM MCh (cmH2O/ml/s), values stated as “0” were excluded due to damage of the lungs.</p