Inhibition of natural killer cells protects the liver against acute injury in the absence of glycine N-methyltransferase

Abstract

Glycine N-methyltransferase (GNMT) catabolizes S-adenosylmethionine (SAMe), the main methyl donor of the body. Patients with cirrhosis show attenuated GNMT expression, which is absent in hepatocellular carcinoma (HCC) samples. GNMT(-/-) mice develop spontaneous steatosis that progresses to steatohepatitis, cirrhosis, and HCC. The liver is highly enriched with innate immune cells and plays a key role in the body's host defense and in the regulation of inflammation. Chronic inflammation is the major hallmark of nonalcoholic steatohepatitis (NASH) progression. The aim of our study was to uncover the molecular mechanisms leading to liver chronic inflammation in the absence of GNMT, focusing on the implication of natural killer (NK) / natural killer T (NKT) cells. We found increased expression of T helper (Th)1- over Th2-related cytokines, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-R2/DR5, and several ligands of NK cells in GNMT(-/-) livers. Interestingly, NK cells from GNMT(-/-) mice were spontaneously activated, expressed more TRAIL, and had strong cytotoxic activity, suggesting their contribution to the proinflammatory environment in the liver. Accordingly, NK cells mediated hypersensitivity to concanavalin A (ConA)-mediated hepatitis in GNMT(-/-) mice. Moreover, GNMT(-/-) mice were hypersensitive to endotoxin-mediated liver injury. NK cell depletion and adoptive transfer of TRAIL(-/-) liver-NK cells protected the liver against lipopolysaccharide (LPS) liver damage.

Conclusion: Our data allow us to conclude that TRAIL-producing NK cells actively contribute to promote a proinflammatory environment at early stages of fatty liver disease, suggesting that this cell compartment may contribute to the progression of NASH.

Figure 1. GNMT deficiency promotes activation and increased cytotoxicity of NK/NKT cells

(A) qRT-PCR of livers from GNMT^−/− and GNMT^+/+ mice. (B) FACS analysis of livers. The graph represents the percentage of NK1.1⁺ cells vs. CD45⁺ cells. Histogram/graph showing NK1.1⁺/CD69⁺ and NK1.1⁺/TRAIL⁺ in livers. (C) Cytotoxicity assay co-culturing liver-derived MNCs with YAC-1 and liver-MACS isolated NK cells with GNMT^−/− hepatocytes at the E:T ratios shown in the figure (D) qRT-PCR of MACS-isolated NK/NKT cells. (E) qRT-PCR of hepatocytes. n=4. *P<0.05; **P<0.01; ***P<0.001 (GNMT^−/− vs. GNMT^+/+). Error bars represent SD.

Figure 2. GNMT deficiency hyper-sensitizes the liver to Concanavalin A-acute hepatitis

(A) ConA increased ALT serum levels. (B) H&E staining (C) TUNEL assay in liver sections (200x) (D) Caspase-3 activity on liver extracts. (E) qRT-PCR (F) Western blot analysis. n=4. *P<0.05; **P<0.01 (GNMT^−/− vs. GNMT^+/+). Error bars represent SD.

Figure 3. GNMT deficiency does not hyper-sensitize the liver to Fas-mediated liver injury

(A) Jo2 injection was lethal for 50% WT animals after 4h whereas no GNMT^−/− mice died. (B) ALT levels on serum (C) H&E staining (200x) (D) TUNEL assay (200x), quantification of TUNEL positive (% vs DAPI⁺ cells) and Caspase3 activity. (E) Western blot analysis (F) qRT-PCR (times vs WT control). n=4. *P<0.05; **P<0.01 (GNMT^−/− vs. GNMT^+/+). Error bars represent SD.

Figure 4. Specific NK cell depletion ameliorates acute liver injury after ConA in GNMT^−/− mice

(A) ASIALO pre-treatment decreased ALT levels, (B) improved liver parenchyma and lower apoptosis (H&E and TUNEL; 200x). (C) qRT-PCR (times vs WT control) (D) ELISA on serum samples. (E) Western blot analysis of livers n=4. *P<0.05; **P<0.01 (GNMT^−/− vs. GNMT^+/+). Error bars represent SD.

Figure 5. GNMT deficiency over-sensitizes the liver to LPS/GalN-mediated liver injury

(A) Increased serum ALT levels, (B) H&E and (C) TUNEL assay (200x) confirmed that LPS/GalN (16μg/kg/800mg/kg) promotes liver injury in GNMT^−/−mice. (D) Western blot analysis (E) qRT-PCR. n=4. *P<0.05; **P<0.01 (GNMT^−/− vs. GNMT^+/+). Error bars represent SD.

Figure 6. Selective depletion of NK cells attenuates strong liver injury after LPS/GalN

(A) Lower ALT serum levels, (B) H&E and TUNEL assay on liver sections confirmed amelioration of liver damage in ASIALO/GNMT^−/− mice (200x). (C) qRT-PCR detecting IFNg, TNF, NOS2, Perforin and IL-4 (times vs WT control). (D) Western blot analysis showed strong p-JNK1/2 and p-c-jun in ASIALO/GNMT^−/− mice 8h after LPS/GalN. GAPDH was used as loading control. n= 4. *P<0.05; **P<0.01 (GNMT^−/− vs. GNMT^+/+). All data are representative of three independent experiments. Error bars represent SD.

Figure 7. Adoptive transfer of liver-TRAIL deficient NK cells protects the liver against LPS/GalN damage in GNMT^−/− mice

ASIALO/GNMT^−/− mice received TRAIL^−/− or GNMT^−/− NK cells isolated from liver. (A) Serum ALT, (B) H&E staining and TUNEL. (C) qRT-PCR (D) Western blot analysis n= 4. *P<0.05; **P<0.01 (GNMT^−/−ASIALO/GNMT^−/−NKs vs. GNMT^−/−ASIALO/TRAIL^−/−NKs); ##p<0.01 (GNMT^−/− vs. GNMT^−/− ASIALO/TRAIL^−/− NKs). Error bars represent SD.

Figure 8. SAMe depletion with NAM protects the liver against LPS/GalN damage in GNMT^−/− mice

GNMT^−/− mice fed with NAM were protected against ConA- and LPS-damage. (A, D) ALT, (B, E) H&E and TUNEL and (C, F) p-JNK western blot. **P<0.01 (GNMT^−/− vs. GNMT^−/−/LPS or ConA); ###p<0.001 (GNMT^−/−/LPS or ConA vs. GNMT^−/−/NAM/LPS or ConA). Error bars represent SD.