Fig 1: 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.
Fig 2: MoKCs play a crucial role in increasing PR3 levels, leading to hepatic inflammation in MASH.a,b, Representative images of immunohistochemical staining for PR3 expression in liver tissue sections from NCD- and FFD-fed mice (a) and healthy controls versus patients with MASH (b). c, Relative mRNA expression of Prnt3 in primary NPCs isolated from NCD- and FFD-fed mice. d, scRNA-seq analysis of NPCs from NCD- and FFD-fed mice, showing the distribution of Prtn3 expression across different cell types. e, Representative co-immunofluorescence images of liver sections from both NCD- and FFD-fed mice stained for CLEC4F (red, indicating Kcs), PR3 (green) and DAPI (blue, indicating nuclei). f, scRNA-seq analysis of EmKCs (Tim4+ and Ccr2−) and MoKCs (Tim4− and Ccr2+) in the Kc pool from NCD- and FFD-fed mice. g, Flow cytometry gating strategy for identifying MoKCs (CD45+, F4/80+, CD11blow, TIM4− and CCR2+) and EmKCs (CD45+, F4/80+, CD11blow, TIM4+ and CCR2−) in liver tissue. h, Quantification of EmKC and MoKC percentages in WT, A1AT KO and A1AT KO mice treated with sivelestat. i, Relative mRNA expression of proinflammatory genes (Tnf, Il1b and Il6) in MoKCs sorted from WT, A1AT KO and A1AT KO mice treated with sivelestat. j, Relative mRNA expression of proinflammatory genes (Tnf, Il1b and Il6) in MoKCs stimulated with CM (CM-control, CM-LPS or CM-LPS + sivelestat). k, Relative mRNA expression levels of proinflammatory genes in ImKCs treated with PR3, LPS and Sivelestat. 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.
Fig 3: The PR3-targeting protein IL-32γ protects against liver inflammation and fibrosis in mice treated with MASH.a, Proteome profiling of a human cytokine array for co-immunoprecipitation of endogenously secreted PR3 in CM from LPS-stimulated (CM-LPS) and nonstimulated (CM-CTRL) THP-1 cells. b, A bar graph indicating the average signal intensities of framed spots on the array blots from a. Each red bracket in the cytokine array analysis indicates the location of interleukin (IL)-32. c, Univariate logistic regression analysis was conducted in a discovery cohort (n = 130) to identify patients ‘at-risk’ from MASH based on the gene expression of PR3-bound cytokines. d, The gene‒disease network for IL32 was examined via data from genome-wide association studies that focused on records with a significance level of P < 1 × 10−6. e, Human macrophage subset gene expression of IL32 based on the Single Cell Portal, Broad Institute. f, A Venn diagram illustrating the ability of PR3 to target the inflammatory cytokine IL-32 and its relationship with SERPINA1 and MASH pathogenesis. g, Relative mRNA expression of proinflammatory genes in primary KCs co-stimulated with LPS and recombinant human IL-32γ (rhIL-32γ; 100 ng/ml) for 6 h. h–j, BWs (h), LWs (i) and LBW ratios (j) of NCD-fed WT mice (n = 5), NCD-fed hIL-32γ Tg mice (n = 5), FFD-fed WT mice (n = 10) and FFD-fed Tg mice (n = 9). k,l, Detection of the serum ALT (k) and cholesterol (l) levels in each group. m, The appearance of freshly collected liver tissues and H&E staining of liver sections from each group. n, IHC staining image of CLEC4F-positive KCs in FFD-fed Tg mice and FFD-fed WT mice. o, BODIPY-stained liver sections from each group. Representative images of BODIPY-stained sections were selected, and BODIPY-positive areas (green, for lipid accumulation) and DAPI-positive areas (blue, for nuclei and normalization) were measured. p, Relative mRNA expression of proinflammatory genes in the hepatic tissues of each group. q, Representative images of Sirius red-stained liver sections from each group. Representative images of whole-body liver sections were analyzed for Sirius red-positive areas. r, Relative mRNA expression of profibrogenic genes in liver sections. 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.
Fig 4: Quintuple deletion of Serpina1a–e exacerbates MASH progression with increased hepatic inflammation and fibrosis.a, Serum A1AT protein levels in A1AT KO and WT mice. b, Serum PR3 levels in NCD-fed WT (n = 5), NCD-fed A1AT KO (n = 5), FFD-fed WT (n = 10) and FFD-fed A1AT KO (n = 10) mice. c, Hepatic tissue mRNA expression of Prtn3 in each group. d–i, BW (d), LW (e), LBW ratio (f) and serum levels of ALT (g), AST (h) and cholesterol (i) in each group. j, Representative images of freshly collected liver tissues and H&E-stained liver sections from each group. k, Representative immunohistochemical staining of CLEC4F-positive KCs in liver tissue sections from each group. Quantification of the CLEC4F-positive area relative to that of WT control mice in both the NCD and FFD groups. l, Relative mRNA expression of proinflammatory genes in hepatic tissues from each group. m, Relative mRNA expression of profibrogenic genes in hepatic tissues from each group. n, Representative images of Sirius red-stained liver tissue sections from each group. The data are presented as the means ± s.d., n ≥ 5; *,#P < 0.05, **,##P < 0.01; ***,###P < 0.001 and ****,####P < 0.0001 versus the control model. ns, not significant.
Fig 5: A1AT supplementation or PR3 inhibition improves steatohepatitis and fibrosis in mice.a–f, BW (a), LW (b), LBW ratio (c), appearance of freshly collected liver tissue samples: treated with Respreeza (d) and sivelestat (e), and serum levels of alanine transferase (f) in the vehicle (NCD), vehicle (FFD), Respreeza (FFD) and sivelestat (FFD) groups. NCD-fed vehicle, n = 5; FFD-fed vehicle, n = 8 and FFD-fed and Respreeza-treated groups, n = 8. FFD-fed vehicle-fed, n = 10 and FFD-fed and sivelestat-treated groups, n = 10. g, Serum PR3 levels in each group using immunoblot. h,i, H&E- and BODIPY-stained liver sections. Each representative image of a BODIPY-stained section was selected and measured to determine the BODIPY-positive area (green, for lipid accumulation) and the 4,6-diamidino-2-phenylindole (DAPI)-positive area (blue, for nuclei and normalization). j, Relative mRNA expression of proinflammatory genes in hepatic tissues from the vehicle (NCD), vehicle (FFD) and Respreeza (FFD) groups. k, Relative mRNA expression of proinflammatory genes in hepatic tissues from the vehicle (FFD) and sivelestat (FFD) groups. l,m, Sirius red-stained liver sections from each group. Each representative image of a whole-body liver section from each group was analyzed to determine the Sirius red-positive area. n,o, Relative expression of profibrogenic genes in hepatic tissue from each group. p, IHC staining image of CLEC4F-positive KCs in each group. 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.
Supplier Page from Abcam for Mouse PR3 ELISA Kit