Journal: Scientific Reports

Article Title: Bile acid metabolites enhance expression of cathelicidin antimicrobial peptide in airway epithelium through activation of the TGR5-ERK1/2 pathway

doi: 10.1038/s41598-024-57251-3

Figure Lengend Snippet: Induction of cathelicidin expression by bile acid metabolites is not mediated through selected nuclear receptors in bronchial epithelium. Expression of ( a ) cathelicidin ( CAMP ) and ( b ) cytochrome P450 family 1 subfamily B member 1 ( CYP1B1 ) genes in BCi cells pretreated for 1 h with CH223191 (2.5 µM) followed by addition of lithocholic acid (LCA; 5 µM) and 3-oxolithocholic acid (3-oxoLCA; 5 µM) in the presence of CH223191 for the next 24 h analyzed by qRT-PCR and presented as a fold change over control (ctrl). n = 3 independent experiments ± SD analyzed by two- and one-way Anova with Sidak’s post-hoc test. The p values are **< 0.01, ***< 0.001, ****< 0.0001 and ns nonsignificant for two-way Anova used to compare CH223191-pretreated cells vs untreated cells within each bile acid treatment group and ## < 0.01, ### < 0.001, #### < 0.0001 for one-way Anova used to compare bile acid treatment to the vehicle control within the CH223191-untreated group. ( c ) Expression of farnesoid X receptor ( FXR ; NR1H4 ) in colonic HT-29 cell line, bronchial BCi and VA10 cell lines, and human primary bronchial/tracheal epithelial cells (HBEpC) presented as a relative expression 2 −ΔCt where ND not detected transcript, n = 3 independent experiments ± SD. ( d ) Expression of the cathelicidin proform (proLL-37) and processing to the LL-37 peptide was analyzed in cell conditioned media after 24 h post treatment of BCi cells by Western blot. Treatment with LCA and 3-oxoLCA was performed with and without 1,25-dihydroxyvitamin D3 (1,25D3; 100 nM) and 4-phenylbutyrate (4-PBA; 2 mM). A combination of 1,25D3 and 4-PBA was used as a positive control for induction of the proLL-37 and the synthetic human LL-37 peptide (sLL-37) was used as a standard control. Expression of GAPDH used as a loading control was analyzed in corresponding cell lysates, n = 3 independent experiments. Protein enriched cell culture conditioned media and corresponding cell lysates were run on separate gels processed in parallel and original blots are presented in Supplementary Fig. . ( e ) Quantification of the band intensity presented as a ratio of ProLL-37 to GAPDH from n = 3 independent experiments ± SD analyzed by one-way Anova with Sidak’s post-hoc test, where p values *< 0.05, **< 0.01, ****< 0.0001 and ns nonsignificant.

Article Snippet: All bile acid metabolites were purchased from Cayman Chem. (Cat. No. 20253; 29545; 29542; 29544), 1,25-dihydroxyvitamin D3 from Sigma (D1530) and 4-PBA (Cat. No. 2682) and CH223191 (Cat. No. 3858) from Tocris.

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot, Positive Control, Cell Culture

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: IL-33/ST2 axis mediates diesel exhaust particles-induced mast cell activation.

doi: 10.1186/s10020-024-01035-y

Figure Lengend Snippet: Fig. 5 IL-33/ST2 axis involved in DEP-induced mast cell migration and type 2 cytokine release. a HMC-1 cells were cultured in the upper chamber of a Transwell system and incubated with CH223191 (CH, 10 μM) or an anti-ST2 blocking antibody (1 μg/ml), followed by stimulation with DEP (10 μg/ml) or IL-33 (10 pg/ml) for 24 h (N = 4). The migrated cells in the lower chamber were harvested and counted. Data are presented as means ± SEM from four independent experiments. *p < 0.05, **p < 0.01 compared to the corresponding vehicle controls; #p < 0.05 compared to the control group. b HMC-1 cells were harvested after stimulation with DEP (0.1–10 μg/ml) for 24 h, and the levels of IL-4, IL-5, and IL-13 were determined by ELISA (N = 4). c HMC-1 cells were stimulated with DEP (10 μg/ml) for 24 h in the presence or absence of an anti-ST2 blocking antibody (1 μg/ml). The levels of IL-4, IL-5, and IL-13 were subsequently determined by ELISA (N = 3). Data are presented as means ± SEM from four (b) and three (c) independent experiments. *p < 0.05; **p < 0.01; ***p < 0.005; ****p < 0.001 compared to DEP treatment alone

Article Snippet: In brief, HMC-1 cells (5 × 105) were seeded in the upper chamber of a TranswellTM plate (pore size = 8 μm; Corning, NY, USA) with culture medium (100 μL) in the presence or absence of CH223191 (Selleckchem, TX, USA) or anti-ST2 (IL1RL1, receptor of IL-33, R&D Systems, Minneapolis, MN, USA) antibody.

Techniques: Migration, Cell Culture, Incubation, Blocking Assay, Control, Enzyme-linked Immunosorbent Assay