Fig. 5: PTX3 is a functionally essential target of METTL3-modulated macrophage activation in allergic asthma.

a Knockdown of Ptx3 in BMDMs using two distinct shRNAs. The M2-associated makers expression in IL-4–stimulated BMDMs was quantified by RT-qPCR (left) (n = 3 cells). The levels of IL-10 secretion were detected by ELISA (right) (n = 6 cells for control group and n = 4 cells for knockdown group). b Western blot analysis of STAT6 and AKT phosphorylation in BMDMs transfected with Ptx3 or Ctrl shRNA, after IL-4 stimulation (n = 3 cells). c RT-qPCR showing M2 activation-associated markers expression in BMDMs from WT and Mettl3 KO mice, with or without Ptx3 knockdown (n = 3 cells for WT group and n = 4 cells for other groups). d Depletion of Ptx3 in CRE–induced allergic asthma model. Serum PTX3 levels were determined by ELISA. e Total and (f) differential BALF cell numbers from experimental animals were analyzed by flow cytometry (n = 5 animals for WT groups and n = 6 animals for KO groups). g Assessment of methacholine-induced AHR in mice (n = 4 animals for WT groups and n = 5 animals for KO groups). h Histopathological changes in the lung tissues were examined by H&E- and PAS-staining (scale bars: 200 μm and 100 μm, respectively). i Calculated inflammation and PAS scores (n = 6 animals). j RT-qPCR (n = 3 cells for WT groups and n = 4 cells for KO groups) and (k) Western blot (n = 3 cells) assays detecting M2-associated markers expression in alveolar macrophages purified from experimental animals. l Flow cytometry analysis of the MFI of the M2 macrophage subpopulation in BALF (n = 4 animals). m Representative composite fluorescent images showing the spatial localization of M2 macrophages (F4/80⁺, red; CD206⁺, green) and PTX3 levels (purple) in lung tissues. Scale bars: 25 μm. Quantification analysis was performed (n = 3 animals). Statistical analysis of the data was performed using two-sided unpaired t test (a right), 1-way ANOVA (d, e, i, k, l, m), and 2-way ANOVA (a left, b, c, f, g, j) followed by either Tukey’s or Sidak’s multiple comparison tests. Data are presented as means ± SEM from one of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; n.s, not significant.