Thus, phosphorylated STAT6 is used as a marker of M2 polarization. which S100A4 exerts its effects. Finally, we inhibit S100a4 in the bleomycin-induced lung fibrosis model by treatment with niclosamide. Our data suggest that S100a4 is produced and secreted by M2 polarized alveolar macrophages and enhances the proliferation and activation of lung fibroblasts. Inhibition of S100a4 might represent a potential therapeutic strategy for pulmonary fibrosis. data using primary macrophages and fibroblasts to investigate the mechanism by which S100a4 exerts its effect. Finally, we inhibit S100a4 with niclosamide in the bleomycin-induced lung fibrosis model. Our data suggest that S100a4 Picrotoxin is produced and secreted by M2 polarized Picrotoxin AMs and enhances the proliferation and activation of lung fibroblasts. Inhibition of S100a4 might represent a potential therapeutic strategy for pulmonary fibrosis. Materials and Methods Preparation and Titration of MHV-68 Virus stocks were grown and quantified by plaque assay as previously described (18). Experiments C57BL/6 mice were purchased from Charles River Laboratories (Sulzfeld, Germany). IFN–R?/? mice on C57BL/6 background were originally obtained from the Jackson Laboratory (Bar Harbor, ME, USA) and subsequently bred and propagated under SPF conditions at the Helmholtz Zentrum Mnchen. Mice were housed in individually ventilated cages during the MHV-68 infection period. Mice (8C12?weeks old) were infected intranasally (i.n.) with 1??105 plaque forming units of MHV-68 diluted in PBS in a total volume of 30?l. Prior to i.n. infection, mice were anesthetized with ketamineCxylazine or with medetomidineCmidazolamCfentanyl. At the predetermined time points, mice were sacrificed by cervical dislocation and subsequently, bronchoalveolar lavage (BAL) was performed and lung tissues were quartered and processed for the following experiments: the left lobe was inflated and fixed in 10% buffered formalin for histological and immunohistochemical examination, and the remaining lobes were stored at ?80C and used for RNA isolation for qRT-PCR to determine gene expression or for preparation of whole lung tissue protein extracts and western blot analysis. To induce pulmonary fibrosis, anesthetized mice (8C12?weeks old) were intratracheally instilled through a MicroSprayer Aerosolizer 20G INTROCAN (Penn Century, Wyndmoor, PA, USA) with 50?l bleomycin (Sigma-Aldrich, Taufkirchen, Germany) in PBS (2?U/kg) or as control, with PBS alone. Mice were analyzed 14?days after bleomycin instillation. To test lung function, mice were anesthetized with ketamine/xylazine, intratracheally intubated through a small incision of the trachea and connected to the flexiVent system (Scireq, Montreal, Canada). Subsequently, mice were sacrificed and lung lobes were snap frozen and stored at ?80C before mRNA isolation and protein determination, and one lobe was filled with 4% paraformaldehyde for histology. Paraffin-embedded sections were stained with hematoxylin and eosin (H&E). Staining intensity was quantified by ImageJ. All animal experiments were in compliance with the German Animal Welfare Act (German Federal Law 8 Abs. Picrotoxin 1 TierSchG), and the protocols were approved by the local Animal Care and Use Committee (District Government of Upper Bavaria; permit number 124-08, 154-13, and 130-14). Bronchoalveolar Lavage Immediately after euthanasia, BAL was conducted via the introduction of a cannula into the trachea. A 1?ml aliquot of ice-cold Dulbeccos phosphate buffered saline (DPBS) was flushed into the airway and gently aspirated TBLR1 a syringe and the tracheal cannula. After the first BAL fluid (BALF) was collected, the BAL continued with seven times of 1 1.5?ml aliquots of PBS until an additional 7?ml of BALF was collected. The initial BALF was then centrifuged at Picrotoxin 1,500?rpm for 5?min at 4C, and the supernatant was collected and decanted into a new 1.5?ml microcentrifuge tube and stored at ?80C for biochemical measurements such as cytokine concentration. The remaining lavage fluid was pooled and centrifuged to remove the supernatant. The sedimented cells together with remnant cell pellets from the first lavage wash were subsequently resuspended in 1?ml PBS. Finally, the number of living cells was counted on a standard hemocytometer in the presence of 0.4% trypan blue (Sigma-Aldrich). Histopathology After BAL harvesting, either the whole lung or the left lobe.