Journal: bioRxiv

Article Title: A Nerve-Fibroblast Axis in Mammalian Lung Fibrosis

doi: 10.1101/2024.09.09.611003

Figure Lengend Snippet: (A-D) Immunofluorescence imaging displayed BrdU (green) and nuclear staining with propidium iodide (PI) (red) in MRC5 human lung fibroblasts. A dose- dependent increase in BrdU incorporation was observed in MRC5 cells stimulated with increasing concentrations of noradrenaline (NA), peaking at 25 µM (D, P = 0.0159). This response was reversed upon co-incubation with terazosin (E, P = 0.0441). (F) MTT assays demonstrated a significant reduction in the number of viable cells in MRC5 human lung fibroblasts and IPF fibroblasts when stimulated with NA and treated with terazosin ( P = 0.0493 and P = 0.0156, respectively). This effect was not observed in normal human lung fibroblasts, indicating that IPF fibroblasts are poised to receive and respond to noradrenergic signals via an α1-AR dependent mechanism. (G-I) Immunofluorescence imaging demonstrated expression of ADRA1D (red) and α-SMA (green), with nuclear staining by DAPI (blue) in human precision-cut lung slices. Following exposure to a fibrotic cocktail, a marked increase in α-SMA (ACTA2) expression was observed in stromal cells adjacent to alveoli, with some cells showing co- expression of ADRA1D (white arrows, G, H). This expression pattern was consistent with trichrome staining and matched ACTA2 expression quantified by PCR analysis (J, P = 0.0236). The introduction of terazosin to the culture media reversed these fibrotic effects (I, J, P = 0.0106). Images were captured at 20x magnification. Data are presented as mean ± SEM or median ± IQR, with statistical analysis performed using Student’s t-test for normally distributed data and Mann-Whitney or Kruskal-Wallis tests with Dunn’s multiple comparisons for non- normally distributed data. * P < 0.05, ** P < 0.01. ADRA1D, α1-adrenoreceptor subtype D; α-SMA, alpha-smooth muscle actin; BrdU, bromodeoxyuridine; DAPI, 4′,6-diamidino-2-phenylindole; FC, fibrotic cocktail; IPF, idiopathic pulmonary fibrosis; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide; NA, noradrenaline; NHLF, normal human lung fibroblast; PCR, polymerase chain reaction; PI, propidium iodide; TZ, terazosin.

Article Snippet: After this incubation, the cells were stained with rat anti-BrdU primary antibody (1:500, BioRad, MCA2483T) for one hour at room temperature.

Techniques: Immunofluorescence, Imaging, Staining, BrdU Incorporation Assay, Incubation, Expressing, MANN-WHITNEY, Polymerase Chain Reaction