Figure 5: Deficiency in PAD4 results in decreased cytoplasts, neutrophils and IL-17.

WT and PAD4−/− mice were sensitized with HDM/LPS and BALFs were collected on day 3. (A) BALF total cell count and (B) leukocyte differential were enumerated (n = 5 mice). (C) Netosis was monitored by BALF DNA levels on day 3 (n = 5 mice), and (D) Western blot for hyper- citrullinated histone H3 (top panel). Ponceau S stain was used as a loading control (bottom panel). (E) Representative flow cytometry plot showing DNA positive PMN and DNA negative cytoplasts in BALFs collected on protocol day 3 after HDM/LPS sensitization from WT and PAD4−/− mice. (F) Percent cytoplasts were enumerated by flow cytometry criteria. (G-K) In addition to the post-sensitization period, the resulting antigen-driven lung inflammation in WT and PAD4−/− mice was determined on protocol day 15 after HDM/LPS sensitization followed by HDM challenge. (G) BALF total cell count and (H) BALF differential count were measured. Lung histology between WT and PAD4−/− mice was evaluated by (I) H&E staining. (J) PAS staining. (K) AHR was measured in anesthetized mice that were mechanically ventilated in the presence of ascending doses of inhaled methacholine. (L) IL-17, and IFN-γ and (M) IL-13, and IL-5. Values represent the mean and error bars SEM (For M and N, error bars represent SD). *p < 0.05, **p < 0.01 using a Student’s t-test.