Fig 2: IPF MPCs colocalize with macrophages in the peripheral region of the fibroblastic focus. Immunohistochemical (IHC) was performed on human IPF lung tissue (n = 10 IPF patient specimens). A: shown are 2 fibroblastic foci. Hematoxylin and eosin (H&E) staining was used to identify the fibroblastic focus and IHC was performed using CD163 (macrophage marker) and SSEA4 and S100A4 (MPC markers) antibodies to assess distribution of macrophages and IPF MPCs in the focus. Asterisk denotes focus core. Bar = 50 µm (left) and 20 µm (middle and right). B: shown are serial 4-µm sections stained for H&E, procollagen, SSEA4, S100A4, and CD163. Shown are two fibroblastic foci. Procollagen expression was used to delineate the myofibroblast-rich focus core. IHC analysis of the foci demonstrated S100A4- and SSEA4-positive cells at the periphery of the focus. CD163-positive cells codistributed with SSEA4- and S100A4-expressing cells at the focus perimeter. Asterisk denotes focus core. Fibroblastic Focus 1: bar = 50 µm (top and middle) and 20 µm (bottom); Fibroblastic Focus 2: bar = 50 µm (top) and 20 µm (bottom). C: IHC analysis of IPF lung tissue for CXCR1 and CXCR2 expression and CD163. Left: dual staining for CXCR1 (brown) and CD163 (red). Shown in the inset is a CD163-positive macrophage (red) with CXCR1 labeling (brown) on the perimeter of the cell. Right: dual staining for CXCR2 (brown) and CD163 (red). Bar = 100 µm (top) and 50 µm (bottom). Asterisk denotes focus core. D: in situ hybridization for IL-8 was performed on IPF lung tissue. Bar = 50 µm (top and bottom left) and 20 µm (bottom right). E: H&E staining and IHC was performed on human control lung tissue (n = 3 control patient specimens) using antibodies to S100A4, SSEA4, and CD163. Bar = 50 µm (H&E) and 20 µm (IHC).

Fig 3: Paracrine function of IL-8 secreted by IPF MPCs. A: CXCR1 and CXCR2 mRNA expression in macrophages. B: macrophage migration in response to fresh medium (control), IPF MPC progeny-derived conditioned medium containing DMSO (CM), IPF MPC progeny-derived conditioned medium containing the CXCR2 inhibitor SB332235 (1 µM) (CM + SB), and IPF MPC progeny-derived conditioned medium containing the CXCR1/2 inhibitor reparixin (100 µM) (CM + R) were analyzed using the QCM Chemotaxis Cell Migration Assay Kit (n = 3). Data are expressed as means ± SE. P values were determined by two-tailed Student’s t-test.