Fig 2: Liver TREM2 immunoreactivity is elevated in patients and mouse models of NASH (A) Liver sections from both patients with NAFL and NASH were immunostained for TREM2 (red), CD68 (green), and DAPI (blue, nucleus stain). Confocal images at × 60 were taken with a Nikon A1 inverted fluorescent microscope, and TREM2+ area was quantified using ImageJ. Individual CD68+ TREM2+ macrophages are depicted at a higher magnification in the inserts. (B) C57BL/6 mice were fed either HF (n = 11) or HFHC (n = 10) diet for 24 weeks. Liver sections were used to immunostain TREM2 (red) and DAPI (blue). Images at × 20 were taken with a Keyence fluorescence microscope (BZ-X800), and TREM2 + area was quantified using image J. (C) Ldlr -/- mice were fed either chow (n = 7) or HFHS diet (n = 9) for 20 weeks and liver sections were immunostained for TREM2 (red) and DAPI. Images at × 20 were taken with a Keyence fluorescence microscope (BZ-X800), and TREM2+ area was quantified using image J. Statistical analysis was performed by unpaired 2-tailed nonparametric Mann-Whitney test (A–C) *p<0.05. Data are shown as mean ± SEM. Scale bar: 50 μm (A) or 20 μm (B–C). Abbreviations: HF, high-fat; HFHC, high-fat, high-cholesterol; HFHS, high-fat, high-sucrose; TREM2, Triggering Receptor Expressed on Myeloid cells 2.

Fig 3: Soluble TREM2 increases lipid droplets and triglyceride content in hepatocytes. (A) BMDMs from WT or ADAM17-deficient mice were cocultured with primary hepatocytes in transwell plates. Cultures were added with BSA or BSA-conjugated palmitate (0.25 mM) for 18 hours in serum-free conditions. Lipid accumulation in hepatocytes was determined by quantification of BODIPY-positive area by ImageJ (n = 4-6). (B) Coculture of ADAM17-deficient BMDMs and hepatocytes stimulated with sTREM2 in transwell plates in the presence of palmitate (0.25mM) for 18 hours. Lipid accumulation in hepatocytes was determined by quantification of BODIPY-positive area by ImageJ (n = 4). (C) Representative images and quantification of BODIPY-positive area or (D) perilipin-2 positive area in mouse primary hepatocytes after treatment with sTREM2 for 18 hours (n = 4) in the presence of palmitate (0.25 mM). (E) Triglyceride levels in mouse primary hepatocytes after treatment with sTREM2 for 18 hours (n = 4) in the presence of palmitate (0.25 mM). Statistical analysis was performed by 1-way ANOVA with Tukey multiple comparisons tests (A–B) or unpaired 2-tailed nonparametric Mann-Whitney test (C–E). Data are shown as mean ± SEM. Abbreviations: BMDM, bone marrow-derived macrophages; BSA, bovine serum albumin; sTREM2, soluble TREM2; TREM2, Triggering Receptor Expressed on Myeloid cells 2; WT, wild-type.

Fig 4: sTREM2 increases lipid droplets by CD36-dependent manner in hepatocytes. (A) Mouse primary hepatocytes were treated with sTREM2 for 18hr and mRNA expression of CD36 normalized to 18S was determined (n = 4). This shows increased CD36 mRNA levels in sTrem2-treated hepatocytes. (B) Representative image and quantification of CD36 protein levels normalized to beta-actin in hepatocytes treated with sTREM2 (n = 4). This shows increased CD36 protein levels in sTrem2-treated hepatocytes. C. Ldlr -/- mice were fed either chow (n = 9) or HFHS diet (n = 10) for 20 weeks, and liver Cd36 mRNA levels were determined by RT-PCR. (D) Mouse primary hepatocytes were pretreated with SSO (25 μM), an inhibitor of CD36. After 30 minutes, cells were treated with sTREM2 with palmitate for 18 hours. Lipid accumulation in hepatocytes was determined by quantification of BODIPY-positive area by ImageJ (n = 4). (E) Working model. Increased ADAM17-mediated shedding of cell surface TREM2 from hepatic macrophages under conditions of Western diet/obesity leads to elevated soluble TREM2 (sTREM2). Elevated levels of sTREM2 increase hepatocyte triglyceride accumulation by increasing CD36-mediated fatty acid uptake, which can aggravate NAFLD progression. Statistical analysis was performed by unpaired two-tailed nonparametric Mann-Whitney test (A–C). Data are shown as mean ± SEM. Abbreviations: HFHC, high-fat, high-cholesterol; HFHS, high-fat, high-sucrose; SSO, sulfosuccinimidyl oleate; sTREM2, soluble TREM2; TREM2, Triggering Receptor Expressed on Myeloid cells 2.

Fig 5: Circulating soluble TREM2 levels are elevated in mice and patients with NASH. (A) Serum samples from NAFLD (n = 15) and NASH (n = 22) patients were analyzed for soluble TREM2 by ELISA. (B), ROC curves for diagnostic accuracy for NASH using serum levels of sTREM2. (C) C57BL/6 mice were fed either HF (n = 11) or HFHC (n = 10) diet for 24 weeks, and liver Adam17 mRNA levels were determined by RNA sequencing. (D) Ldlr -/- mice were fed either chow (n = 9) or HFHS diet (n = 10) for 20 weeks, and liver Adam17 mRNA levels were determined by RT-PCR. (E) Serum samples from mice fed chow (n =9) or HFHS diet (n = 6) were analyzed for soluble TREM2 by ELISA. Statistical analysis was performed by unpaired 2-tailed nonparametric Mann-Whitney test. (A, C–E). Data are shown as mean ± SEM. (F) Gallbladder bile samples from mice fed chow, HF, or HFHC diet for 3 months were analyzed for sTREM2 by ELISA. Statistical analysis was performed by unpaired t-test, and data are shown as mean ± SD. Abbreviations: HF, high-fat; HFHC, high-fat, high-cholesterol; HFHS, high-fat, high-sucrose; sTREM2, soluble TREM2; TREM2, Triggering Receptor Expressed on Myeloid cells 2.