Loss of macrophage TSC1 exacerbates sterile inflammatory liver injury through inhibiting the AKT/MST1/NRF2 signaling pathway

Abstract

Tuberous sclerosis complex 1 (TSC1) plays important roles in regulating innate immunity. However, the precise role of TSC1 in macrophages in the regulation of oxidative stress response and hepatic inflammation in liver ischemia/reperfusion injury (I/R) remains unknown. In a mouse model of liver I/R injury, deletion of myeloid-specific TSC1 inhibited AKT and MST1 phosphorylation, and decreased NRF2 accumulation, whereas activated TLR4/NF-κB pathway, leading to increased hepatic inflammation. Adoptive transfer of AKT- or MST1-overexpressing macrophages, or Keap1 disruption in myeloid-specific TSC1-knockout mice promoted NRF2 activation but reduced TLR4 activity and mitigated I/R-induced liver inflammation. Mechanistically, TSC1 in macrophages promoted AKT and MST1 phosphorylation, and protected NRF2 from Keap1-mediated ubiquitination. Furthermore, overexpression AKT or MST1 in TSC1-knockout macrophages upregulated NRF2 expression, downregulated TLR4/NF-κB, resulting in reduced inflammatory factors, ROS and inflammatory cytokine-mediated hepatocyte apoptosis. Strikingly, TSC1 induction in NRF2-deficient macrophages failed to reverse the TLR4/NF-κB activity and production of pro-inflammatory factors. Conclusions: Macrophage TSC1 promoted the activation of the AKT/MST1 signaling pathway, increased NRF2 levels via reducing Keap1-mediated ubiquitination, and modulated oxidative stress-driven inflammatory responses in liver I/R injury. Our findings underscore the critical role of macrophage TSC1 as a novel regulator of innate immunity and imply the therapeutic potential for the treatment of sterile liver inflammation in transplant recipients. Schematic illustration of macrophage TSC1-mediated AKT/MST1/NRF2 signaling pathway in I/R-triggered liver inflammation. Macrophage TSC1 can be activated in I/R-stressed livers. TSC1 activation promotes phosphorylation of AKT and MST1, which in turn increases NRF2 expression and inhibits ROS production and TLR4/NF-κB activation, resulting in reduced hepatocellular apoptosis in I/R-triggered liver injury.

Schematic illustration of macrophage TSC1-mediated AKT/MST1/NRF2 signaling pathway in I/R-triggered liver inflammation. Macrophage TSC1 can be activated in I/R-stressed livers. TSC1 activation promotes phosphorylation of AKT and MST1, which in turn increases NRF2 expression and inhibits ROS production and TLR4/NF-κB activation, resulting in reduced hepatocellular apoptosis in I/R-triggered liver injury.

Fig. 1. TSC1 expression is increased in liver macrophages and negatively correlated with I/R-stressed liver injury in patients.

A, B Western blot analysis of the relative protein level of TSC1 in macrophage in liver specimens from patients undergoing orthotopic liver transplantation (OLT) or partial hepatectomy (PHY). C, D Thirty-five human OLTs were divided into low ratio of post-OLT TSC1/GAPDH group (Low TSC1: n = 18) and high ratio of post-OLT TSC1/GAPDH group (High TSC1: n = 17) by using post-OLT TSC1/GAPDH ratio = 0.784 as threshold. Thirty-five human PHY were divided into low ratio of post-PHY TSC1/GAPDH group (Low TSC1: n = 18) and high ratio of post-PHY TSC1/GAPDH group (High TSC1: n = 17) by using post-PHY TSC1/GAPDH ratio = 1.194 as threshold. sALT values in both Low TSC1 and High TSC1 groups in OLT recipients or patients undergoing PHY at POD1. E, F The ratio of post-OLT TSC1/GAPDH and post-PHY TSC1/GAPDH both correlated negatively with sALT at POD1. G, H Histology (H&E staining) and TUNEL staining of liver specimens from patients undergoing OLT or partial hepatectomy, Scale bars = 100 μm (H&E staining), Scale bars = 50 μm (TUNEL staining). *p < 0.05.

Fig. 2. Myeloid-specific TSC1 deficiency exacerbates hepatocellular damage in I/R-induced liver injury.

A Western-blot analysis of TSC1, p-MST1 and MST1 protein expression in liver macrophages from I/R-stressed livers from mice subjected to 90 min of partial liver warm ischemia, followed by 6 h, 12 h or 24 h of reperfusion. B Western-blot analysis of TSC1 in macrophages during I/R. C Mice were subjected to 90 min of partial liver warm ischemia, followed by 6 h of reperfusion. Representative histological staining (H&E) of ischemic liver tissue (n = 4–6/group), Scale bars = 250 μm. D Liver damage, evaluated by Suzuki’s score. ***p < 0.001. E Hepatocellular function, as assessed by serum ALT levels (IU/L). The results are expressed as the Mean ± SD (n = 4–6 samples/group). ***p < 0.001. F Liver neutrophil accumulation, as determined by MPO activity (U/g). The results are expressed as the Mean ± SD (representative of 4–6 mice/group). **p < 0.01. G Quantitation of ROS-sensing dye DHE staining, MDA, and GSH activities were assessed in ischemic liver tissues. *p < 0.05, **p < 0.01.

Fig. 3. Myeloid-specific TSC1 deficiency increases hepatocellular apoptosis in I/R-triggered livers.

A, B Liver apoptosis analyzed by TUNEL staining, Scale bars = 200 μm. The results were scored semi-quantitatively by averaging the number of apoptotic cells (Mean ± SD) per field at 400× magnification. The results are representative of 4–6 mice/group. ***p < 0.001. C Western blot analysis of BCL-2, BCL-xL and Cleaved Caspase 3. β-actin served as an internal control. The data are representative of three experiments. D Caspase-3 activity. The results are expressed as the Mean ± SD (n = 4–6 samples/group). ***p < 0.001.

Fig. 4. Myeloid-specific TSC1 deficiency increases macrophage/neutrophil trafficking, inhibits AKT/MST1/NRF2 activation, and induces TLR4 signaling in I/R-induced liver injury.

A Liver CD11b⁺ macrophages and Ly6G⁺ neutrophils were detected by immunohistochemistry. Results were scored semi-quantitatively by averaging the number of positively-stained cells (Mean ± SD)/field at 400× magnification. Representative of 4–6 mice/group, Scale bars = 150 μm. ***p < 0.001. B Quantitative RT-PCR-assisted detection of TNF-α, IL-1β, IL-6 and TGF-β expression. Mean ± SD (n = 4–6 samples/group). **p < 0.01. C Western blot analysis of AKT, phosphorylated AKT, MST1, phosphorylated MST1, NRF2 and TLR4. β-actin served as an internal control. Data are representative of three experiments. D Cells were stained with fluorochrome-conjugated anti-F4/80 or -CD11b. F4/80 and CD11b double-positive cells were identified as infiltrating macrophages. E Western blot analysis of AKT, p-AKT, MST1, p-MST1 and NRF2 in infiltrating macrophages. β-actin served as an internal control. Data are representative of three experiments.

Fig. 5. AKT is required for the regulation of MST1/NRF2 in myeloid TSC1-deficient livers in response to I/R stress.

Bone marrow-derived macrophages (BMMs) from TSC1^M-KO mice were transfected with lentivirus expressing AKT (Lv-AKT) or GFP control (Lv-GFP) and adoptively transferred into TSC1^M-KO mice. A Representative H&E and TUNEL staining of liver sections (n = 4–6 mice/group), Scale bars = 250 μm (H&E), Scale bars = 200 μm (TUNEL). B Liver damage, evaluated by Suzuki’s score. ***p < 0.001. C The number of apoptotic cells per field at 400× magnification. The results are representative of 4–6 mice/group. ***p < 0.001. D The serum ALT levels from the indicated groups. ***p < 0.001. E Representative immunohistochemistry staining and quantification of CD11b⁺ macrophages and Ly6G⁺ neutrophils in liver sections (n = 4–6 mice/group), Scale bars = 150 μm. **p < 0.01, ***p < 0.001. F Western blot analysis of p-AKT, p-MST1, MST1, NRF2, and TLR4. β-actin served as an internal control. Data are representative of three experiments. G Quantitative RT-PCR-assisted detection of TNF-α, IL-1β, IL-6 and TGF-β expression. Mean ± SD (n = 4–6 samples/group). **p < 0.01.

Fig. 6. MST1 overexpression ameliorates myeloid-specific TSC1 deficiency-mediated liver damage in I/R-induced liver injury.

BMMs from TSC1^M-KO mice were transfected with lentivirus expressing MST1 (Lv-MST1) or GFP control (Lv-GFP) and adoptively transferred into TSC1^M-KO mice. A The serum ALT levels from the indicated groups (n = 4–6 mice/group). ***p < 0.001. B Representative H&E staining and Suzuki’s score of liver sections (n = 4–6 mice/group), Scale bars = 250 μm. ***p < 0.001. C Representative immunohistochemistry staining and quantification of Ly6G⁺ neutrophils in liver sections (n = 4–6 mice/group), Scale bars = 150 μm. **p < 0.01. D Western blot analysis of p-MST1, NRF2, and TLR4. β-actin served as an internal control. Data are representative of three experiments. E Quantitative RT-PCR-assisted detection of TNF-α, IL-1β, IL-6 and TGF-β expression. Mean ± SD (n = 4–6 samples/group). **p < 0.01.

Fig. 7. Silencing of Keap1 ameliorates TSC1 deficiency-mediated liver damage in I/R-induced liver injury.

TSC1^M-KO mice were injected via the tail vein with a mannose-mediated Keap1 siRNA or NS siRNA at 4 h prior to ischemia. A Representative histological staining (H&E, original magnification x100) (4–6 mice/group), Scale bars = 250 μm. B Liver damage, as evaluated by Suzuki’s score. ***p < 0.001. C Hepatocellular function, as assessed by serum ALT levels (IU/L). Results are expressed as the Mean ± SD (n = 4–6 samples/group). ***p < 0.001. D Liver CD11b⁺ macrophages and (E) Ly6G⁺ neutrophils were detected by immunohistochemistry. Results were scored semi-quantitatively by averaging the number of positively-stained cells (Mean ± SD)/field at 400×magnification. Representative of 4–6 mice/group. ***p < 0.001. F Western blot analysis of Keap1, NRF2, HMGB1, NF-κB and TLR4. β-actin served as an internal control. Data are representative of three experiments. G Quantitation of ROS-sensing dye DHE staining, MDA, and GSH activities were assessed in ischemic liver tissues. *p < 0.05, **p < 0.01.

Fig. 8. AKT is crucial for TSC1-mediated MST1/NRF2 activation in macrophages in vitro.

Bone marrow-derived macrophages (BMMs) were isolated from TSC1^FL/FL and TSC1^M-KO mice and pretreated with Lv-AKT or Lv-GFP controls before LPS stimulation for 6 h. A Immunoprecipitation analysis of AKT and MST1 in macrophages after LPS stimulation. Representative of three experiments. B Immunofluorescence localization of DAPI (blue), p-AKT (red) and p-MST1 (green) in LPS-stimulated BMMs, Scale bars = 50 μm. C Western blot analysis of phosphorylated AKT, MST1, phosphorylated MST1, HMGB1, NF-κBp65 and TLR4 in LPS-stimulated BMMs. β-actin served as an internal control. D Quantitative RT-PCR-assisted detection of TNF-α, IL-1β, IL-6 and TGF-β in LPS-stimulated BMMs. Mean ± SD (n = 3–4 samples/group). **p < 0.01. E Western blot analysis of TSC1, AKT, phosphorylated AKT, MST1, phosphorylated MST1, NRF2, and NF-κBp65 in LPS-stimulated BMMs followed by Over-TSC1 or Vector-pretreatment. β-actin served as an internal control. Data are representative of three experiments.

Fig. 9. Macrophage TSC1-mediated MST1 regulates TLR4/NF-κB activity and hepatocyte apoptosis in vitro.

Bone marrow-derived macrophages (BMMs) were isolated from TSC1^FL/FL and TSC1^M-KO mice and pretreated with Lv-MST1 or Lv-GFP controls before LPS stimulation for 6 h. A Western blot analysis of phosphorylated MST1, HMGB1, TLR4, and NF-κBp65 in LPS-stimulated BMMs. β-actin served as an internal control. B Quantitative RT-PCR detection of TNF-α, IL-1β, IL-6 and TGF-β in LPS-stimulated BMMs. Mean ± SD (n = 3–4 samples/group). **p < 0.01. C ROS production was detected by Carboxy-H2DFFDA in LPS-stimulated BMMs from TSC1^M-KO mice. Quantification of ROS-producing BMMs (green), Scale bars = 20 μm. **p < 0.01. D, E Apoptotic hepatocytes from the BMM/hepatocyte coculture system were evaluated by flow cytometry. Representative of three separate experiments. **p < 0.01.

Fig. 10. NRF2 is essential for TSC1-mediated immune regulation in macrophages in vitro.

Bone marrow-derived macrophages (BMMs) were isolated from NRF2^FL/FL and NRF2^M-KO mice and pretreated with Lv-TSC1 or Lv-GFP controls before LPS stimulation for 6 h. A ROS production was detected by Carboxy-H2DFFDA in LPS-stimulated BMMs from TSC1^M-KO mice, Scale bars = 20 μm. B Quantification of ROS-producing BMMs (green). **p < 0.01. C Western blot analysis of TSC1, NRF2, HMGB1, NF-κB and TLR4. β-actin served as an internal control. Data are representative of three experiments. D Quantitative RT-PCR-assisted detection of TNF-α, IL-1β, IL-6 and TGF-β in LPS-stimulated BMMs. Mean ± SD (n = 3–4 samples/group). *p < 0.05, **p < 0.01. E BMMs were transfected with CRISPR-TSC1 activation vector (1 and 3 µg, respectively). Immunoprecipitation analysis of Keap1 and NRF2 in macrophages after LPS stimulation. F Immunoblot analysis of NRF2 ubiquitination in total lysates (bottom) and anti-IgG or anti-NRF2 immunoprecipitates (IP, top) of WT and TSC1-KO BMMs, probed with anti-ubiquitin (α-Ub) and antibodies to NRF2, TSC1, and GAPDH.