Fig. 8: PR3-dependent proteolytic degradation and cleavage of IL-32γ contribute to the activation of KCs and HSCs.

a, Relative mRNA expression of proinflammatory genes in primary KCs from IL-32γ Tg mice co-stimulated with LPS and A1AT KO HCM or WT HCM. b, Immunoblot analysis of PR3-mediated cleavage and degradation of rhIL-32γ in the presence of phosphate-buffered saline (PBS) at different time points at 37 °C. c, Proteolytic process of IL-32γ in CM from IL-32γ (WT)- and IL-32γ (V104A)-overexpressing ImKCs stimulated with LPS, as determined via an immunoblot assay. Each CM was concentrated via a concentrator (4,000 rpm for 25 min at 4 °C). d, Relative mRNA expression of proinflammatory genes in ImKCs treated with CM from LPS- and sivelestat-treated EV ImKCs, IL-32γ (WT) ImKCs and IL-32γ (V104) ImKCs. e, A schematic diagram illustrating the hypothesis that PR3-mediated IL-32γ (WT) cleavage induces immune responses, whereas the anti-inflammatory effect of IL-32γ (V104A) is maintained. f, Immunoblot analysis of the cleavage of rhIL-32γ by PR3 in the presence of sivelestat. rhIL-32γ was preincubated with PR3 for 5 min at 37 °C, and the reaction was stopped with a serine protease inhibitor. g, Relative mRNA expression of proinflammatory genes in ImKCs treated with rhIL-32γ, PR3 and sivelestat. h,i, Relative mRNA expression of M1 and M2 markers in primary KCs co-treated with LPS or the full-length (FL)-rhIL-32γ (100 ng/ml), C-terminal (100 ng/ml) or N-terminal (100 ng/ml) domain of IL-32γ. j, Detection of TNF in the CM of primary KCs co-treated with LPS, FL-rhIL-32γ (100 ng/ml), the C-terminal domain (100 ng/ml) or the N-terminal domain (100 ng/ml) of IL-32γ. k, Immunoblot analysis of p-JNK, JNK, p-ERK, ERK, p-IκB-α, IκB-α, p-p65, p65 and actin protein levels in ImKCs stimulated with LPS with or without the C-terminal cleaved form of IL-32γ. l, Graphical figure showing that the cleaved c-terminal domain of IL-32γ by PR3 contributes to the M1 polarization of KCs. m, Relative mRNA expression of profibrogenic genes in primary HSCs in the quiescent state (after 1 day of culture) and activated state (after 3 days of culture) stimulated with FL-rhIL-32γ (100 ng/ml) and the C-terminus (100 ng/ml). n, The graphical representation illustrates that the cleavage of IL-32γ fails to prevent the activation of HSCs. The data are presented as the means ± s.d.; *^,#P < 0.05, **^,##P < 0.01, ***^,###P < 0.001 and ****^,####P < 0.0001 versus the control model. ns, not significant.