Investigating the Fibrogenic Role of Citrullinated Vimentin

To evaluate the fibrogenetic role of Cit-Vim, C3H/HeOuJ TLR4 WT and C3H/HeJ TLR4 MUT (a spontaneous mutation in the TLR4 gene, Tlr4^lps-d, The Jackson Laboratory) mice were treated by intratracheal instillation (IT) once to Sal, native Vim, or Cit-Vim (2 mg/kg). In other experiments, PAD2^−/− mice (provided by S. A. Coonrod), Akt1^−/−Lyz2-cre mice with a conditional deletion of Akt1 in macrophages (46 (link)), and TLR4 MUT and WT mice in parallel were given once with CdCl₂ (0.16 mg/kg, Sigma-Aldrich) and/or CB (5.0 mg/kg, Sigma-Aldrich). For BALF, three instillations of 1 ml of phosphate-buffered saline (PBS) were performed for each mouse and the differential number of cells in BALF was counted according to standard hematological procedures. Lung tissues were dried and acid-hydrolyzed (6 N HCl at 95°C for 20 hours), and collagen deposition was assessed by checking hydroxyproline concentration as per the manufacturer’s protocols (Chondrex).