Myeloid Notch1 deficiency activates the RhoA/ROCK pathway and aggravates hepatocellular damage in mouse ischemic livers

Abstract

Notch signaling plays an emerging role in the regulation of immune cell development and function during inflammatory response. Activation of the ras homolog gene family member A/Rho-associated protein kinase (ROCK) pathway promotes leukocyte accumulation in tissue injury. However, it remains unknown whether Notch signaling regulates ras homolog gene family member A/ROCK-mediated immune responses in liver ischemia and reperfusion (IR) injury. This study investigated intracellular signaling pathways regulated by Notch receptors in the IR-stressed liver and in vitro. In a mouse model of IR-induced liver inflammatory injury, we found that mice with myeloid-specific Notch1 knockout showed aggravated hepatocellular damage, with increased serum alanine aminotransferase levels, hepatocellular apoptosis, macrophage/neutrophil trafficking, and proinflammatory mediators compared to Notch1-proficient controls. Unlike in the controls, myeloid Notch1 ablation diminished hairy and enhancer of split-1 (Hes1) and augmented c-Jun N-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1), JNK, ROCK1, and phosphatase and tensin homolog (PTEN) activation in ischemic livers. Disruption of JSAP1 in myeloid-specific Notch1 knockout livers improved hepatocellular function and reduced JNK, ROCK1, PTEN, and toll-like receptor 4 activation. Moreover, ROCK1 knockdown inhibited PTEN and promoted Akt, leading to depressed toll-like receptor 4. In parallel in vitro studies, transfection of lentivirus-expressing Notch1 intracellular domain promoted Hes1 and inhibited JSAP1 in lipopolysaccharide-stimulated bone marrow-derived macrophages. Hes1 deletion enhanced JSAP1/JNK activation, whereas clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9-mediated JSAP1 knockout diminished ROCK1/PTEN and toll-like receptor 4 signaling.

Conclusion: Myeloid Notch1 deficiency activates the ras homolog gene family member A/ROCK pathway and exacerbates hepatocellular injury by inhibiting transcriptional repressor Hes1 and inducing scaffold protein JSAP1 in IR-triggered liver inflammation; our findings underscore the crucial role of the Notch-Hes1 axis as a novel regulator of innate immunity-mediated inflammation and imply the therapeutic potential for the management of organ IR injury in transplant recipients. (Hepatology 2018;67:1041-1055).

Figure 1. Myeloid-specific Notch1 deficiency aggravates IR-induced hepatocellular damage

Mice were subjected to 90min of partial liver warm ischemia, followed by 6h or 24h of reperfusion. (A) The Notch1 expression was detected in hepatocytes and liver macrophages by Western blot assay. Representative of three experiments. (B) Liver function in serum samples was evaluated by sALT levels (IU/L). Results expressed as mean±SD (n=4-6 samples/group). **p<0.01. (C) Representative histological staining (H&E) of ischemic liver tissue. Results representative of 4-6 mice/group; original magnification ×100. Liver damage, evaluated by Suzuki's histological score. **p<0.01. (D) Liver neutrophil accumulation, analyzed by MPO activity (U/g). Mean±SD (n=4-6 samples/group). *p<0.05.

Figure 2. Myeloid-specific Notch1 deficiency increases macrophage/neutrophil infiltration and proinflammatory mediators in liver IRI

By 6h of reperfusion after 90min of ischemia, liver macrophages and neutrophils were detected by immunofluorescence and immunohistochemistry staining using mAbs against mouse CD11b⁺ and Ly6G in Notch1^FL/FL ( ) and Notch1^M-KO ( ) mice. (A) Immunofluorescence staining of CD11b⁺ macrophages in ischemic livers. Quantification of CD11b⁺ macrophages per high power field. Results scored semi-quantitatively by averaging number of positively-stained cells (mean±SD)/field at 200×magnification. Representative of 4-6 mice/group. *p<0.05. (B) Quantitative RT-PCR-assisted detection of TNF-α, IL-1β, and MCP-1 in mouse livers. Each column represents the mean±SD (n=3-4 samples/group). *p<0.05, **p<0.01. (C) Immunohistochemistry staining of Ly6G+ neutrophils in ischemic livers. Quantification of Ly6G+ neutrophils per high power field (original magnification ×200). Representative of 4-6 mice/group. **p<0.01.

Figure 3. Myeloid-specific Notch1 deficiency depresses Hes1 but induces RhoA/ROCK activation in IR-stressed liver

(A) Quantitative RT-PCR-assisted detection of mRNA coding for Hes1 and RhoA in mouse livers at 6h of reperfusion followed by 90min of ischemia. Each column represents the mean±SD (n=3-4 samples/group). **p<0.01. (B) Western-assisted analysis and relative density ratio of NICD, Hes1, JSAP1, p-JNK, and ROCK1. Representative of three experiments. *p<0.05, **p<0.01. (C) ELISA analysis of IL-1β, MCP-1, and IL-6 levels in animal serum. Mean±SD (n=3-4 samples/group), *p<0.05, **p<0.01.

Figure 4. Myeloid-specific Notch1 deficiency increases hepatocellular apoptosis in IR-stressed liver

(A) Liver apoptosis by TUNEL staining in mouse liver at 6h of reperfusion followed by 90min of ischemia. Results scored semi-quantitatively by averaging the number of apoptotic cells (mean±SD) per field at 200× magnification. Representative of 4-6 mice/group, *p<0.05. (B) Caspase-3 activity. Mean±SD; n=4-6 samples/group. **p<0.01. (C) Western-assisted analysis and relative density ratio of PTEN, p-Akt, and TLR4 Representative of three experiments. **p<0.01. (C) ELISA analysis of TNF-α levels in animal serum. Mean±SD (n=3-4 samples/group), **p<0.01.

Figure 5. Activation of RhoA/ROCK pathway triggers innate immune response and IR-induced inflammatory injury in myeloid Notch1-deficient liver

The Notch1^M-KO and Notch1^FL/FL mice were injected via tail vein with nonspecific (NS) control siRNAs ( ) or ROCK1 siRNA ( ) (2 mg/kg) mixed with mannose-conjugated polymers at 4h prior to ischemia. (A) The severity of liver IRI was evaluated by the Suzuki's histological grading at 6h of reperfusion followed by 90min of ischemia. *p<0.05. (B) Hepatocellular function was evaluated by sALT levels (IU/L). Results expressed as mean±SD (n=4-6 samples/group). **p<0.01. (C) Immunofluorescence staining of CD11b⁺ macrophages in ischemic livers. Quantification of CD11b⁺ macrophages per high power field. Results scored semi-quantitatively by averaging number of positively-stained cells (mean±SD)/field at 200×magnification. Representative of 4-6 mice/group. **p<0.01. (D) Immunohistochemistry staining of Ly6G+ neutrophils in ischemic livers. Quantification of Ly6G+ neutrophils per high power field (original magnification ×200). Representative of 4-6 mice/group. *p<0.05. (E) Western blots analysis and relative density ratio of PTEN, p-Akt, and TLR4. Representative of three experiments. *p<0.05, **p<0.01. (F) Quantitative RT-PCR-assisted detection of mRNA coding for TNF-α, IL-1β, and MCP-1. Each column represents the mean±SD (n=3-4 samples/group). *p<0.05, **p<0.01.

Figure 6. Myeloid-specific Notch1 deficiency activates RhoA/ROCK pathway via a JSAP1-dependent manner in IR-stressed livers

The Notch1^M-KO and Notch1^FL/FL mice were injected via tail vein with nonspecific (NS) control siRNAs ( ) or JSAP1 siRNA ( ) (2 mg/kg) mixed with mannose-conjugated polymers at 4h prior to ischemia. (A) Representative histological staining (H&E) of ischemic liver tissue at 6h of reperfusion followed by 90min of ischemia. Results representative of 4-6 mice/group; original magnification ×100. The severity of liver IRI was evaluated by the Suzuki's histological grading. **p<0.01. (B) Hepatocellular function was evaluated by sALT levels (IU/L). Results expressed as mean±SD (n=4-6 samples/group). **p<0.01. (C) ELISA analysis of TNF-α levels in animal serum. Mean±SD (n=3-4 samples/group), **p<0.01. (D) Western blots analysis and relative density ratio of p-JNK, ROCK1, PTEN, TLR4, and cleaved caspase-3. Representative of three experiments. *p<0.05, **p<0.01. (E) Quantitative RT-PCR-assisted detection of mRNA coding for RhoA, IL-1β, and MCP-1. Each column represents the mean±SD (n=3-4 samples/group). **p<0.01.

Figure 7. Myeloid Notch1-Hes1 axis is crucial in the regulation of JSAP1-dependent RhoA/ROCK activation in macrophages

(A) BMMs from Notch1^M-KO mice were transfected with the lentivirus expressing NICD (LV-pSIN-NICD) or the control vector (LV-control) followed by LPS (100 ng/ml) stimulation. Western-assisted analysis and relative density ratio of Hes1, and JSAP1. Representative of three experiments. **p<0.01. (B) BMMs from Notch1^FL/FL mice were transfected with the lentiviral-mediated CRISPR/Cas9-mediated Hes1 knockout (LV-Hes1 KO) or the LentiCRISPRv2 vector without gRNA sequence control (LV-control) followed by LPS (100 ng/ml) stimulation. Western-assisted analysis and relative density ratio of Hes1, JSAP1, ROCK1, and PTEN. Representative of three experiments. **p<0.01. (C) ELISA-assisted production of TNF-α in cell culture supernatants. Mean±SD (n=3-4 samples/group). **p< 0.01. (D) Quantitative RT-PCR-assisted detection of mRNA coding for IL-1β and MCP-1. Each column represents mean±SD (n=3-4 samples/group). *p<0.05. (E) BMMs from Notch1^M-KO mice were transfected with the lentiviral-mediated CRISPR/Cas9-mediated JSAP1 knockout (LV-JSAP1 KO) or the LentiCRISPRv2 vector without gRNA sequence control (LV-control) followed by LPS (100 ng/ml) stimulation. Western-assisted analysis and relative density ratio of JSAP1, ROCK1, PTEN, TLR4, and p-IκBα. Representative of three experiments. **p<0.01. (F) ROS production was detected by Carboxy-H2DFFDA in LPS-stimulated BMMs from Notch1^M-KO mice. Positive green fluorescent-labeled cells were counted blindly in 10 HPF/section (×200). Quantification of ROS-producing BMMs (green) per high power field (×200). **p<0.01. (G) Quantitative RT-PCR-assisted detection of mRNA coding for TNF-α, IL-1β and MCP-1. Each column represents mean±SD (n=3-4 samples/group). *p<0.05, **p<0.01. ( ) Cells only; ( ) LV-pSin-NICD; ( ) LPS + LV-Hes1 KO; ( ) LPS + LV-control; ( ) LPS + LV-JSAP1 KO.

Figure 8. Schematic illustration of myeloid Notch1 signaling in the regulation of innate immune response in IR-triggered liver inflammation

Notch1 can be activated in IR-stressed livers. Upon ligand binding, Notch1 is cleaved by γ-secretase leading to a release of the intracellular domain (NICD), which translocates into the nucleus and forms a complex with the CSL DNA-binding protein and activates its target gene Hes1. Induction of Hes1 inhibits JNK binding protein JSAP1-mediated ROCK1 activation. Blockade of ROCK1 reduces PTEN and augments Akt activity, leading to suppressed TLR4 signaling in liver IRI. In addition, the Notch-Hes1 axis inhibits JSAP1-dependent ROCK1 and caspase-3 activity, resulting in reduced hepatocellular apoptosis/necrosis in IR-triggered liver inflammation.