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, 7 (9), e2390

Activation of Necroptosis in Human and Experimental Cholestasis

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Activation of Necroptosis in Human and Experimental Cholestasis

Marta B Afonso et al. Cell Death Dis.

Abstract

Cholestasis encompasses liver injury and inflammation. Necroptosis, a necrotic cell death pathway regulated by receptor-interacting protein (RIP) 3, may mediate cell death and inflammation in the liver. We aimed to investigate the role of necroptosis in mediating deleterious processes associated with cholestatic liver disease. Hallmarks of necroptosis were evaluated in liver biopsies of primary biliary cholangitis (PBC) patients and in wild-type and RIP3-deficient (RIP3-/-) mice subjected to common bile duct ligation (BDL). The functional link between RIP3, heme oxygenase-1 (HO-1) and antioxidant response was investigated in vivo after BDL and in vitro. We demonstrate increased RIP3 expression and mixed lineage kinase domain-like protein (MLKL) phosphorylation in liver samples of human PBC patients, coincident with thioflavin T labeling, suggesting activation of necroptosis. BDL resulted in evident hallmarks of necroptosis, concomitant with progressive bile duct hyperplasia, multifocal necrosis, fibrosis and inflammation. MLKL phosphorylation was increased and insoluble aggregates of RIP3, MLKL and RIP1 formed in BLD liver tissue samples. Furthermore, RIP3 deficiency blocked BDL-induced necroinflammation at 3 and 14 days post-BDL. Serum hepatic enzymes, fibrogenic liver gene expression and oxidative stress decreased in RIP3-/- mice at 3 days after BDL. However, at 14 days, cholestasis aggravated and fibrosis was not halted. RIP3 deficiency further associated with increased hepatic expression of HO-1 and accumulation of iron in BDL mice. The functional link between HO-1 activity and bile acid toxicity was established in RIP3-deficient primary hepatocytes. Necroptosis is triggered in PBC patients and mediates hepatic necroinflammation in BDL-induced acute cholestasis. Targeting necroptosis may represent a therapeutic strategy for acute cholestasis, although complementary approaches may be required to control progression of chronic cholestatic liver disease.

Figures

Figure 1
Figure 1
Necroptosis is activated in the liver of patients with PBC. (a) Representative H&E-stained sections of liver from control subjects (n=5) and PBC patients (n=5); scale bar, 100 μm (left). Representative RIP3 and MLKL immunofluorescence (red) in liver from control and PBC patients; scale bar, 10 μm (right). (b) Representative immunoperoxidase staining for p-MLKL in human liver samples. Scale bar, 50 μm (left). Representative p-MLKL immunofluorescence (red) and thioflavin T staining (green) in liver from control and PBC patients. Scale bar, 10 μm (right). Nuclei were counterstained with Hoechst 33258 (blue). Histograms show the quantification of RIP3, total MLKL, p-MLKL and thioflavin T mean fluorescence intensity and quantification of colocalization area of thioflavin T and p-MLKL, as described in Materials and Methods section. Quantification was performed in at least eight high-power fields per liver sample. Data are expressed as mean±S.E.M. fold change. §P<0.05 from Control
Figure 2
Figure 2
Necroptosis is activated in the liver of mice after BDL. C57BL/6N mice were subjected to sham or BDL surgical procedures and killed at days 3 and 14. (a) Representative H&E and Massons' Trichrome-stained liver sections. (b) qRT-PCR analysis of RIP3 and MLKL in mouse liver. (c) Immunoblotting and densitometry of total RIP3, p-MLKL, MLKL and RIP1. (d) RIP3, MLKL and RIP1 in insoluble and soluble fractions of liver whole-cell lysates. Blots of RIP3, MLKL and RIP1 were normalized to endogenous β-actin, whereas p-MLKL was normalized to MLKL. Representative immunoblots are shown. Results are expressed as mean±s.e.m. fold change of 7–10 individual mice. §P<0.05 and *P<0.01 from respective sham-operated mice; P<0.05 and P<0.01 from respective BDL day 3. (e) Representative in vitro RIP3 kinase activity from sham and BDL mice
Figure 3
Figure 3
RIP3 deficiency ameliorates hepatic necroinflammation in the BDL murine model. C57BL/6N WT and RIP3−/− mice were subjected to sham or BDL surgical procedures and killed at days 3 and 14. (a) Representative images of H&E-stained liver sections (top). Necrosis and inflammatory cell infiltration was scored as described in Materials and Methods section (bottom). Results are expressed as mean±s.e.m. fold change of 4–5 individual mice. (b) qRT-PCR analysis of MIP-2, IL-1β and TLR4 in mouse liver. Results are expressed as mean±S.E.M. fold change of 7–10 individual mice. §P<0.05 and *P<0.01 from sham-operated mice; P<0.05 and P<0.01 from BDL WT mice at respective time-point
Figure 4
Figure 4
Deletion of RIP3 does not improve BDL-induced fibrosis and apoptosis. C57BL/6N WT and RIP3−/− mice were subjected to sham or BDL surgical procedures and killed at days 3 and 14. (a) qRT-PCR analysis of α-SMA, collagen-1α1 and TGFβ in mouse liver. Results are expressed as mean±S.E.M. fold change of 7–10 individual mice. (b) Representative images of Masson's Trichrome-stained liver sections (top). Periportal fibrosis and bile duct hyperplasia were scored as described in Materials and Methods section (bottom). Results are expressed as mean±s.e.m. fold change of 4–5 individual mice. (c) TUNEL staining of liver tissue sections. Nuclei were counterstained with Hoechst 33258 (blue). Scale bar, 30 μm (left). Histograms show the quantification of TUNEL-positive cells/mm2 (right). Results are expressed as mean±S.E.M. fold change of 4–5 individual mice. (d) Caspase-3/7 activity assay. §P<0.05 and *P<0.01 from sham-operated mice; P<0.05 from BDL WT mice at respective time-point
Figure 5
Figure 5
RIP3−/− BDL mice display differential hepatic expression of HO-1, iron accumulation and oxidative stress. C57BL/6N WT and RIP3−/− mice were subjected to sham or BDL surgical procedures and killed at days 3 and 14. (a) Immunoblotting and densitometry RIP3 and HO-1. Blots were normalized to endogenous β-actin. Representative immunoblots are shown. (b) qRT-PCR analysis of HO-1 and iNOS in mouse liver. (c) Labile iron content in whole-liver lysates measured as described in Materials and Methods section. Results are expressed as mean±s.e.m. fold change of 7–10 individual mice (top). Representative images of Perls' Prussian Blue-stained liver sections. Iron accumulation score was performed as described in Materials and Methods section (bottom). Results are expressed as mean±s.e.m. fold change of 4–5 individual mice. (d) qRT-PCR analysis of hepcidin, FtH and FtL in mouse liver. (e) Malondialdehyde content in whole-liver lysates as a surrogate of lipid peroxidation measured as described in Materials and Methods section (left). Fluorescence intensity from whole-cell lysates stained with the fluorescent probe H2DCFDA was measured (right). Values are corrected with total protein content. Results are expressed as mean±S.E.M. fold change of 7–10 individual mice. §P<0.05 and *P<0.01 from sham-operated mice; P<0.05 and P<0.01 from BDL WT mice at respective time-point
Figure 6
Figure 6
HO-1 is involved in iron accumulation and cytotoxic effects of GCDCA in RIP3-deficient primary mouse hepatocytes. Primary mouse hepatocytes isolated from WT and RIP3−/− C57BL/6N mice were treated with CoPP (10 μM), ZnPP (15 μM) or vehicle control. After 1 h, cells were exposed to either GCDCA (50 μM) or vehicle control. After 24 h, cells were harvested for total iron or immunoblotting. Cell death assays were performed after 48 h. (a) Total iron levels in whole cells were measured as described in Materials and Methods section. Values were normalized with total protein concentration. (b) Percentage of general cell death in primary mouse hepatocytes as assessed by LDH activity assay. (c) Apoptotic cells detected by Hoechst staining. Results are expressed as percentage of apoptotic cells (bottom). Representative images of untreated WT and RIP3−/− primary hepatocytes and incubated with GCDCA, GCDCA plus CoPP or GCDCA plus ZnPP are shown. Scale bar, 30 μM. (d) Primary mouse hepatocytes isolated from WT and RIP3−/− C57BL/6N mice were treated with Nec-1 (100 μM) or vehicle control. After 1 h, cells were exposed to GCDCA (50 μM) for 48 h. Percentage of general cell death was as assessed by the LDH activity assay. Results are expressed as mean±S.E.M. fold change or percentage from three independent cultures from each genotype. §P<0.05 and *P<0.01 from Control; P<0.05 and P<0.01 from respective control

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