Incomplete Deletion of IL-4Rα by LysMCre Reveals Distinct Subsets of M2 Macrophages Controlling Inflammation and Fibrosis in Chronic Schistosomiasis
Figure 5
A population of inflammatory IL-4Rα-expressing myeloid cells resists LysMCre-mediated deletion.
BALB/c, IL-4Rαflox/Δ, and IL-4Rαflox/ΔLysMCre mice were injected i.p. with 2 ml thioglycollate 4 d prior to harvest or were left untreated (naïve). Peritoneal cells were harvested from each group, stimulated for 30 min with 20 ng/ml IL-4 (black outline), and compared to unstimulated cells (solid gray). IL-4Rα function was assessed by IL-4-induced phosphorylation of STAT6 using flow cytometry. A, D. Gating strategy for lymphocytes and F4/80hi CD11bhi macrophages. Detection of pSTAT6 in lymphocytes (B, E) and macrophages (C, F). Each histogram peak represents an individual mouse (n = 2–6 for 2 independent experiments). G. DNA was isolated from F4/80hi CD11bhi macrophages FACS sorted from naïve and thioglycollate-treated IL-4Rαflox/flox, IL-4Rαflox/Δ, IL-4Rαflox/ΔLysMCre and IL-4RαΔ/Δ mice. Rearrangement of the Il4rα locus was measured using PCR to compare the presence of wild-type (WT) and knockout (KO) Il4rα alleles. Quantification of band intensity is shown in the right panels. Aggregate intensity of WT product plus KO product was normalized to 100 percent for each sample. H. In a separate experiment, CD11b+ F4/80+ macrophages were sorted from the same naïve or thioglycollate-elicited peritoneal cells. Lyz2 gene expression was found to be lower in thioglycollate-elicited macrophages than naïve macrophages (n = 2–5 for 2 independent experiments). Data shown are mean ±SEM and represent two independent experiments (*p<0.05).