Figure 3: PTEN deficient macrophages display an enhanced M2- like phenotype in vitro and during fibrosis.
Alveolar macrophages (AMs) were isolated from the lungs of myeloid PTEN knockout mice and wild-type littermates and stimulated with IL-4 (10 ng/ml), IL-13 (10 ng/ml) or both in combination. (a) AMs were stimulated with IL-4 and IL-13 for the indicated time points and phosphorylation of AKT and GSK3β was detected by Western Blot, which were increased in PTEN deficient macrophages. (b) IL-4/IL-13 stimulation overnight led to increased Arginase I (Arg1) expression in the macrophages lacking PTEN. (c–e) AMs were stimulated with IL-4 or IL-13 o/n, RNA was isolated and analysed by qPCR. The M2 markers Resistin like alpha (Retnla, FIZZ1), Arg1 and Chitinase-like 3 (Chil3, YM1) were determined. The Data were analysed by 2-way ANOVA, n = 4, **p < 0.01, ***p < 0.001, ****p < 0.0001. (f) Western Blot analysis of phospho-AKT, phospho-GSK3β, AKT and β-Actin of macrophages isolated from single cell suspension of lungs of wild-types and myeloid PTEN deficient mice 14d post BLM treatment and control mice. (g) Total lung of BLM treated mice was homogenized at d7 and RNA was isolated. Via qPCR the expression of the genes stabilin1 (stab1), retnla, arg1 and chil3 was measured. For statistical analysis the Mann Whitney test was performed, n ≥ 4, *p < 0.05. Expression levels were normalized to HPRT as housekeeping gene. Data are shown as fold values of BLM treated wild-type (light bars) and PTENMyKO (grey bars) mice relative to treatment-naive mice.
