TREM2 Regulates the Removal of Apoptotic Cells and Inflammatory Processes during the Progression of NAFLD

Abstract

Nonalcoholic fatty liver disease (NAFLD) is the most common liver pathology worldwide. In mice and humans, NAFLD progression is characterized by the appearance of TREM2-expressing macrophages in the liver. However, their mechanistic contributions to disease progression have not been completely elucidated. Here, we show that TREM2⁺ macrophages prevent the generation of a pro-inflammatory response elicited by LPS-laden lipoproteins in vitro. Further, Trem2 expression regulates bone-marrow-derived macrophages (BMDMs) and Kupffer cell capacity to phagocyte apoptotic cells in vitro, which is dependent on CD14 activation. In line with this, loss of Trem2 resulted in an increased pro-inflammatory response, which ultimately aggravated liver fibrosis in murine models of NAFLD. Similarly, in a human NAFLD cohort, plasma levels of TREM2 were increased and hepatic TREM2 expression was correlated with higher levels of liver triglycerides and the acquisition of a fibrotic gene signature. Altogether, our results suggest that TREM2⁺ macrophages have a protective function during the progression of NAFLD, as they are involved in the processing of pro-inflammatory lipoproteins and phagocytosis of apoptotic cells and, thereby, are critical contributors for the re-establishment of liver homeostasis.

Keywords: TREM2; fibrosis; inflammation; lipoproteins; macrophages; metabolic-associated fatty liver disease (MAFLD); nonalcoholic fatty liver disease (NAFLD); nonalcoholic steatohepatitis (NASH); phagocytosis.

Figure 1

LPS-containing TRLs elicit a macrophage-dependent pro-inflammatory response in the liver (a) LPS levels were measured in serum and TRL particles isolated from healthy and obese patients (n = 6). (b–e) Mice were pre-treated or not with clodronate to deplete macrophages and then injected without or with LPS-containing TRL particles. Liver tissue was harvested 4 h after injection. Hepatic gene expression of Tnfa (b), Il1b (c), Il6 (d) and Trem2 (e) (n = 5–6). Data are shown as mean + SEM. Statistically significant differences between groups (p < 0.05) were determined by two-way ANOVA and are indicated as p-value (a) or as different letters (b–e).

Figure 2

Trem2 controls LPS- and TRL-induced hepatic pro-inflammatory response (a–c) WT (Trem2^+/+) and Trem2^−/− mice were injected with TRL particles. (a) Western blot of JNK1 and JNK2 as well as phosphorylated JNK1 and JNK2 and (b) its quantification (n = 4). (c) Hepatic gene expression of Tnfa, Il1b and Il6 (n = 7). (d) KC from WT (Trem2^+/+) and Trem2^−/−mice were treated with LPS and secretion of TNFα, IL1β and IL6 into the media was measured after 4 and 18 h (n = 4). (e) Gene expression of Lpl, Cd36 and Lipa in WT (Trem2^+/+) and Trem2^−/− BMDMs. (f) Uptake of DiD-labeled TRL particles into WT (Trem2^+/+) and Trem2^−/− macrophages (n = 9–10). Data are shown as mean + SEM. Statistically significant differences between groups (p < 0.05) were determined by Student’s t-test (a–e) or Mann–Whitney test (f) and are indicated as p-value.

Figure 3

Trem2 regulates KC-dependent uptake of apoptotic cells (a) KCs were isolated from WT (Trem2^+/+) and Trem2^−/− mice and incubated with labeled aHs at 4 °C (binding control) and 37 °C (binding+uptake); (left panel): gating strategy, (right panel): quantification of phagocyting macrophages (n = 4–5). (b) BMDMs were isolated from WT (Trem2^+/+) and Trem2^−/− mice and incubated with labeled apoptotic hepatocytes at 4 °C (binding control) and at 37 °C (uptake); (left panel): gating strategy (right panel): quantification of phagocyting macrophages (n = 5–6). (c) Hepatic macrophages were isolated from WT (Trem2^+/+) and Trem2^−/− mice, pre-treated with control (IgG) or anti-CD14 antibody (aCD14), stimulated with LPS for 20 h and incubated with labeled aHs. Phagocyting macrophages were quantified (n = 8). Data are shown as mean + SEM. Statistically significant differences between groups (p < 0.05) were determined by Mann–Whitney test (a,b) and Student’s t-test (c) and are indicated as p-value.

Figure 4

Loss of Trem2 results in the accumulation of apoptotic cells and aggravates liver damage. (a) Western Blotdepicting CASPASE 3 and cleaved CASPASE 3 levels as a marker for hepatic apoptosis and (b) quantification of cleaved CASPASE 3 expression (n = 4) and (c) gene expression of Bax performed on liver samples isolated from of WT (Trem2^+/+) and Trem2^−/− mice (n = 4). WT (Trem2^+/+) and Trem2^−/− mice were fed with an HFD and (d) the frequency of hepatic apoptotic cells detected by Annexin V staining (n = 6), and (e) frequency of differentially polarized macrophage populations is shown (n = 6). (f–m). WT (Trem2^+/+) and Trem2^−/− mice as well as WT (Trem2^+/+) and Trem2^−/− mice deficient for Apoe were fed a HFD for 16 weeks. (f) Body weight, (g) liver weight, (h) liver triglyceride (TG) levels, (i) H&E staining of liver sections (scale bar = 200 µm), (j) plasma ALT levels, hepatic gene expression of (k) Tgfb1 and (l) Col1a1 (n = 5–8) and (m) Sirius red staining of liver sections (scale bar = 200 µm). Data are shown as mean + SEM. Statistically significant differences between groups (p < 0.05) were determined by Student’s t-test (b,c,f–h,j–l) and Mann–Whitney test (d,e) and are indicated as p-value.

Figure 5

TREM2 in human NAFLD. (a) Levels of soluble TREM2 in human healthy controls or NASH patients (n = 19–21). (b–d) Correlation of hepatic TREM2 expression with (b) plasma ALT levels, (c) hepatic COL1A1 and (d) liver triglyceride (TG) levels in a human cohort (n= 120). (a) Data are shown as mean + SEM. Statistically significant differences between groups (p < 0.05) were determined by Student’s t-test and are indicated as p-value. (b–d) Correlation is given as Pearson R.