Figure 4

GC frass-induced PAR-2 mRNA and cytokine production from BMDC. A. Bone marrow was isolated from BALB/c mice and cultured in the presence of GM-CSF for 6 days. Cells (5 × 10⁶) were cultured in the presence or absence of GC frass (1 μg/ml) for 4 h. Cells were extracted in TRIzol, RNA was synthesized and converted to cDNA. Quantitative real time PCR was performed. PAR-2 was normalized to β-actin and levels are expressed as fold-increase over control (mean ± SEM for 4 separate experiments; *p < 0.001). B-D. Bone marrow was isolated from BALB/c and PAR-2-deficient mice and was cultured in the presence of GM-CSF for 6 days. Cells (1 × 10⁶) were cultured in the presence or absence of GC frass (1 μg/ml) or Pam3Cys (100 ng/ml) for 18 h. Cell supernatants were harvested, clarified and analyzed for cytokine expression by ELISA. In all cases data are expressed as means ± SEM for 4 separate experiments. B. IL-6 expression (*p < 0.001, **p < 0.05). C. IL-23 expression (*p < 0.001; **p = 0.002). D. TNFα expression (compared to PBS *p < 0.001; compared to GC frass **p = 0.023).