Fig 2: Amphiregulin Induces Pericyte Differentiation by Releasing Bioactive TGF-ß via Integrin-aV(A and B) Primary lung pericytes were cultured in the presence or absence of 100 ng/ml Amphiregulin (A), then incubated with latent TGF-ß in the presence and absence of PLC? inhibitor U-73122 (B) and analyzed for LAP binding by flow cytometry.(C–F) After 24 h of treatment, the release of bioactive TGF-ß (upper panel), as well as their differentiation into myofibroblasts (lower panels), was determined in the presence or absence of inhibitors for the EGFR (Gefitinib) (C), PLC? inhibitor (U-73122) (D), integrin-aV (CWHM-12 and its inactive control enantiomer CWHM-96) (E), or TGF-ß-R (ALK5i) (F).(G) The induction of aSMA in treated pericytes was also evaluated at the protein level by western blot analysis.(H) Differentiation of primary lung pericytes into myofibroblasts following o/n co-culture with (left graph) or o/n exposure to supernatants derived from (right graph) alveolar macrophages isolated from infected or uninfected WT or Areg-/- mice.All data are representative of at least two independent experiments except for (B)–(D) (mean ± SEM); results for preparations from individual mice are shown as dots. See also Figure S4.

Fig 3: r TGF-ß Restores Tissue Repair in Areg-/- Mice(A–C) WT or Areg-/- mice were either left uninfected or infected with N. brasiliensis. On days 1, 2, and 3 pi, mice were treated with 5 µg of rTGFß or left untreated. (A) Oxygen saturation in the blood at different dpi, (B) number of red blood cells in the BAL, and (C) extravasation of Evans blue into the alveolar space at 4 dpi were evaluated.(D–G) Egfrflox/flox or Egfrflox/flox × Pdgfrb-cre mice were either left uninfected or infected with N. brasiliensis. On days 1, 2, and 3 pi, mice were treated with 5 µg of either rAREG or rTGFß or left untreated. (D) Oxygen saturation in the blood at different dpi, (E) number of red blood cells in the BAL, (F) extravasation of Evans blue into the alveolar space, and (G) expression of the aSMA and collagen a1 types I and III were evaluated at 4 dpi.Data represent mean ± SEM; results for individual mice are shown as dots. See also Figure S5.

Fig 4: Amphiregulin Contributes to the Restoration of Blood Barrier and Lung FunctionWT and Areg-/- mice were either left uninfected or infected with N. brasiliensis and either injected with 5 µg of rAREG at days 1, 2, and 3 post infection or left untreated.(A) Representative H&E staining and histological analysis of lung tissue at different dpi (days post infection).(B) Oxygen saturation in the blood at different dpi.(C) Number of red blood cells in the BAL (bronchoalveolar lavage).(D) Extravasation of Evans blue into the alveolar space as a marker of vascular permeability.(E) mRNA (Acta2) and protein expression of the aSMA at 4 dpi were evaluated by qRT-PCR and WB.All data are representative of at least two independent experiments (mean ± SEM); results for individual mice are shown as dots. See also Figure S1.