Fig 1.
Spns2 expression in human macrophages.
A, Expression and subcellular localization of Spns2 in alveolar macrophages. Spns2 (rabbit antibody) was labeled in red, beta-actin in green, blue was staining of nuclei. Spns2 was localized to plasma membrane (arrowhead), nucleus (arrow), and cytoplasm. B, Representative confocal images of Spns2 (goat antibody, green) in COPD vs. non-COPD alveolar macrophages, captured for quantitative analysis. For the highly intense Spns2 immunofluorescence in the given COPD sample not become saturated, the laser settings had to be set as low as the immunofluorescence in the non-COPD sample was nearly invisible for naked eyes. Scale bars in A and B are in micrometers. C, Significant increase of Spns2 immunofluorescence in alveolar macrophages of smokers/COPD patients (n = 5) compared to healthy control (C, n = 8, p<0.01) and non-COPD non-smoker transplant patients (Tx, n = 4, p<0.01). D, Cigarette smoke extract induced significant increase (**, p<0.01) of Spns2 immunofluorescence in primary alveolar macrophages obtained from non-smokers (n = 6). E, Cigarette smoke extract induced significant increase (***, p<0.001, 5 experiments) of Spns2 immunofluorescence in THP-1 macrophages. F, Cigarette smoke extract induced significant increase (*, p<0.05, 3 experiments) of Spns2 mRNAs in THP-1 macrophages.
Fig 2.
Human bronchial epithelial cells abundantly express SPHK1, SPHK2 and Spns2.
A, SPHK1 (red). B, SPHK2 (red). C, Spns2 (red, LifeSpan BioSciences rabbit polyclonal antibody). Green in A-C was beta-actin. D, E, Spns2 (green, Santa Cruz goat polyclonal antibody) was expressed in cilia, and colocalized with SPHK1 (red, yellow being merged color of red and green) in the cytoplasm. F, a negative staining control incubated with conjugated antibodies alone. Blue in A-F was DAPI. Arrows in C, D, E indicate Spns2 expression in cilia. Scale bars are in micrometers. Images are representative of bronchial epithelial cells obtained from 3 different non-smoking donors.
Fig 3.
Cigarette smoke extract induced decrease of Spns2 expression in epithelial cells.
A, Representative confocal images of Spns2 in primary cultures of human nasal epithelial cells (hNAEc), cigarette smoke extract-treated (CS) vs. control (C). B, Measurement of Spns2 mean fluorescence intensity (MFI) in hNAEc; **, p = 0.001 significant reduction in CS-treated cells, data pooled from 2 experiments. C, Representative confocal images of Spns2 in 16HBE cell line, treated with CS vs. control. D, Measurement of Spns2 mean fluorescence intensity (MFI) in 16HBE cells; ***, p<0.001 significant reduction in CS-treated cells, data pooled from 2 experiments. Red: Spns2; Green: cyto-keratin (epithelial-specific marker) in A, and β-actin in C. Scale bar = 20μm.
Fig 4.
Defective phagocytosis in lung macrophages of cigarette smoked mice was associated with a down-regulated expression of Spns2 in bronchial epithelia.
A, Representative confocal images of Spns2 in lung tissue of control and cigarette smoke (CS)-exposed mice, and a negative control staining (NEG). Spns2 was stained in red, nuclei in blue. Scale bars are in micrometers. B, Significant decrease of Spns2 (MFI, mean fluorescence intensity) in bronchial epithelia in CS-exposed vs. control mice (p<0.001, pooled data from 6 animals per group). C, Cigarette smoke exposure induced a significant decrease of efferocytosis (p<0.05, n = 6 per group) in lung macrophages of CS-exposed vs. control mice, and a nearly significant trend of reduced NTHi phagocytosis (p = 0.063). D, Correlation between NTHi phagocytic activity of pulmonary macrophages in individual mice and expression of Spns2 in their bronchial epithelia (pooled data from 12 mice, r = 0.685; p = 0.014 by Spearman’s rho test).