Fig. 9: Bi-specific neutrophil protease inhibitors can suppress activation of all IL-36 subfamily cytokines simultaneously.

HeLa^IL-36R-SEAP cells were stimulated with a combination of recombinant human IL-36 proteins (IL-36α + β + γ, all 500 pM final) pre-incubated for 2 h at 37 °C with a titration of PMA-activated neutrophil degranulate (or control unstimulated neutrophil S/N 1:4) in the presence or absence of the mono-specific (z-EPF-cmk or z-API-cmk) or bi-specific (z-EPF-API-cmk or z-API-EPF-cmk) cathepsin G/elastase peptide inhibitors (all at 5 μM). After 24 h, cytokine concentrations in the culture supernatants were determined by ELISA. Results shown are representative of at least three independent experiments. Error bars represent the mean ±SEM of triplicate determinations from a representative experiment. Asterisk(s) indicate significance levels, ***p < .0001, **p < .001, *p < .1, by Student’s t test. ND not detected, indicates where cytokine production was below the detection threshold of the assay