Disruption of myeloid-specific Notch1 aggravates IR-induced liver injury and increases macrophage accumulation and proinflammatory mediators in IR-stressed liver. The myeloid-specific Notch1-deficient (Notch1M − KO), Notch1-proficient (Notch1FL/FL), and WT mice were subjected to 90min of warm ischemia followed by 6h of reperfusion. The liver macrophages (Kupffer cells) were isolated from WT groups. First, we found that IR stress-induced increased nuclear Notch intracellular domain (NICD) in Kupffer cells (Fig. 1A). The Notch1M − KO livers showed severe edema, sinusoidal congestion, and necrosis compared to the Notch1FL/FL livers, which showed mild to moderate edema, sinusoidal congestion, and narrow hepatocellular necrosis (Fig. 1B). The liver damage was evaluated by Suzuki’s histological grading of liver IRI. The hepatocellular functions are measured by the serum ALT (sALT) levels (IU/L). Myeloid Notch1 deficiency significantly augmented sALT levels at 6h post-liver reperfusion in the Notch1M − KO mice compared to the Notch1FL/FL controls (Fig. 1C). Moreover, the Notch1M − KO ischemic livers showed increased accumulation of CD11b+ macrophages (Fig. 1D), accompanied by elevated mRNA levels coding for TNF-α, IL-1β, CCL-2, and CXCL-10 in ischemic livers (Fig. 1E), compared to the Notch1FL/FL controls. These results suggest that the Notch1 pathway plays a critical role in IR stress-induced liver inflammation and injury.
Disruption of myeloid-specific Notch1 exacerbates IR-triggered hepatocyte death.
The myeloid-specific Notch1-deficient (Notch1M − KO) and Notch1-proficient (Notch1FL/FL) mice were subjected to 90min of warm ischemia followed by 6h of reperfusion. Immunofluorescence staining revealed increased TUNEL+ hepatocytes in Notch1M − KO mice compared to Notch1FL/FL group after IR injury (Fig. 2A). The expression of Bax and cleaved-caspase3 were remarkably increased in Notch1M − KO mice, accompanied by the down-regulated level of Bcl-2 in ischemic livers (Fig. 2B). Similarly, immunohistochemistry staining of cleaved caspase-3 showed increased positive cells counts in Notch1M − KO mice (Fig. 2C). In addition, knockout of myeloid Notch1 in mice down-regulated level of β-catenin, while up-regulated the TRAF6, p-TAK1, p-P65, RIPK3, and p-MLKL expression (Fig. 2D), along with the enhanced level of RIPK3 using immunohistochemistry staining after IR injury (Fig. 2E). These results indicate that the disruption of myeloid-specific Notch1 exacerbates IR-triggered hepatocyte death.
β-catenin is essential for the macrophage Notch1-mediated inflammatory response in liver IRI. To investigate the role of β-catenin in macrophage Notch1-mediated immune regulation in IR-stressed liver, an adoptive transfer of lentivirus β-catenin-modified macrophages was constructed. Interestingly, overexpression of β-catenin in the Notch1M − KO mice alleviated IR-induced liver damage evidenced by decreased Suzuki’s histological score (Fig. 3A) and sALT and sAST levels (Fig. 3B), compared to the control group. Moreover, lentivirus β-catenin treatment in the Notch1M − KO ischemic livers reduced CD11b+ macrophage (Fig. 3C) and Ly6G+ neutrophil (Fig. 3D) accumulation. The mRNA levels of TNF-α, IL-1β, CCL-2, and CXCL-10 in Notch1M − KO ischemic livers were also inhibited after the overexpression of β-catenin in macrophage (Fig. 7E). Unlike the control group, lentivirus β-catenin treatment reduced TRAF6, p-TAK1, and p-P65 expression (Fig. 7F) in the Notch1M − KO livers. These results demonstrate β-catenin is a key regulator of Notch1-mediated inflammatory response in IR-stress livers.
Myeloid Notch1 signaling controls immune regulation and hepatocyte death in a RIPK3-dependent manner in IR-stressed liver. Having found that the RIPK3 functions were augmented in Notch1M − KO mice in liver IRI, we then examined whether RIPK3 is essential in liver inflammation and cellular injury in Notch1-mediated IR-stressed livers. We disrupted RIPK3 in Notch1M − KO livers with an in vivo mannose-mediated RIPK3 siRNA delivery system that specifically delivers to macrophages by expressing a mannose-specific membrane receptor as previously described(20). Unlike the administration of non-specific (NS) siRNA in the Notch1M − KO mice, the knockdown of RIPK3 significantly (p < 0.05) decreased IR-induced liver damage (Fig. 4A) and the sALT and sAST levels (Fig. 4B) in the Notch1M − KO mice. RIPK3 siRNA treatment in the Notch1M − KO livers reduced CD11b+ macrophage (Fig. 4C) and Ly6G neutrophil (Fig. 4D) accumulation. Moreover, RIPK3 knockdown decreased mRNA levels of TNF-α, IL-1β, CCL-2, and CXCL-10 in Notch1 myeloid knockout mice (Fig. 4E). Notably, RIPK3 siRNA treatment alleviated hepatocyte apoptosis evidenced by decreased TUNEL+ cells in IR-stressed livers compared to the NS siRNA-treated livers (Fig. 4F). These results demonstrate RIPK3 plays a critical role in the Notch1-mediated immune regulation and hepatocyte apoptosis in IRI livers.
β-catenin interacts with NICD and mediates TAK1 activation in macrophages. Having demonstrated that β-catenin is a key regulator of Notch1-mediated inflammatory response in IR-stress livers, we then analyzed putative crosstalk between β-catenin and the TAK1 pathway in macrophages. Interestingly, Immunofluorescence staining revealed that β-catenin co-localized with NICD in LPS-stimulated BMMs (Fig. 5A). The co-immunoprecipitation assay revealed β-catenin could directly bound to endogenous NICD in BMMs after LPS stimulation (Fig. 5B). To further confirm that β-catenin is required for the crosstalk between Notch1 and TAK1-mediated innate immune responses in vitro, we introduced CRISPR/Cas9. Strikingly, CRISPR/Cas9-mediated β-catenin activation reduced TRAF6, p-TAK1, and p-P65 expression (Fig. 5C) in macrophages from Notch1M − KO mice. Similarly, CRISPR/Cas9-mediated β-catenin KO increased TRAF6, p-TAK1, and p-P65 expression (Fig. 5D) in macrophages from Notch1FL/FL mice. These results indicate that β-catenin interacts with NICD and controls the crosstalk between Notch1 and TAK1-mediated innate immune responses.
TRAF6 is essential for the Notch1-mediated inflammatory response and ROS generation in LPS-stimulated macrophages. To further confirm the role of TRAF6 in the regulation of macrophage Notch1-mediated immune responses in IR-stressed liver, BMMs were isolated from the Notch1FL/FL and Notch1M − KO mice and transfected with CRISPR/Cas9-mediated TRAF6 activation or TRAF6 KO vector. Indeed, CRISPR/Cas9-mediated TRAF6 activation increased p-TAK1 and p-P65 expression in Notch1FL/FL macrophages (Fig. 6A), along with augmented mRNA levels coding for TNF-α, IL-1β, CCL-2, and CXCL-10 (Fig. 6B) in Notch1FL/FL macrophages after LPS-stimulation. ROS production presented by Carboxy-H2DFFDA showed enhanced ROS generation in CRISPR-TRAF6 activation macrophages (Fig. 6C). Moreover, CRISPR/Cas9-mediated TRAF6 KO diminished p-TAK1 and p-P65 expression (Fig. 6D). The mRNA levels coding for TNF-α, IL-1β, CCL-2, and CXCL-10 were also reduced (Fig. 6E), accompanied by down-regulated ROS production in Notch1M − KO macrophages after LPS-stimulation (Fig. 6F). These results indicate that TRAF6 is critically involved in activating TAK1-mediated inflammatory responses.
Macrophage Notch1 deficiency-mediated TRAF6 enhances stress-induced hepatocyte necroptosis via modulating RIPK3 signaling activation. We then asked how macrophage TRAF6 may regulate hepatocyte necroptosis under inflammatory conditions. Using a co-culture system with LPS-stimulated CRISPR-TRAF6 KO BMMs from the Notch1M − KO mice and primary hepatocytes supplemented with H2O2 (Fig. 7A), We found decreased TNF-α release from LPS-stimulated Notch1M − KO BMMs after transfection with a CRISPR-TRAF6 KO vector compared to the control groups (Fig. 7B). Moreover, our results showed that macrophage TRAF6 KO markedly inhibited LDH release from stressed hepatocytes by H2O2 in the coculture supernatant (Fig. 7C). unlike the control groups, macrophage TRAF6 KO decreased hepatocyte p-MLKL, RIPK3 expression (Fig. 7D). Strikingly, Immunofluorescence staining revealed reduced TUNEL+ hepatocytes after co-culture with the CRISPR-TRAF6 KO BMMs but not the control cells (Fig. 7E). These results indicate that macrophage TRAF6 diminishes hepatocyte necroptosis by regulating RIPK3-pMLKL activation in response to inflammatory responses.