Inhibition of Triggering Receptor Expressed on Myeloid Cells 1 Ameliorates Inflammation and Macrophage and Neutrophil Activation in Alcoholic Liver Disease in Mice

Abstract

Alcoholic liver disease (ALD) is characterized by macrophage and neutrophil leukocyte recruitment and activation in the liver. Damage- and pathogen-associated molecular patterns contribute to a self-perpetuating proinflammatory state in ALD. Triggering receptor expressed on myeloid cells 1 (TREM-1) is a surface receptor that amplifies inflammation induced by toll-like receptors (TLRs) and is expressed on neutrophils and monocytes/macrophages. We hypothesized that TREM-1 signaling contributes to proinflammatory pathway activation in ALD. Using an in vivo ALD model in mice, we tested the effects of ligand-independent TREM-1 inhibitory peptides that were formulated into human high-density lipoprotein (HDL)-mimicking complexes GF9-HDL and GA/E31-HDL. As revealed in vitro, macrophages endocytosed these rationally designed complexes through scavenger receptors. A 5-week alcohol feeding with the Lieber-DeCarli diet in mice resulted in increased serum alanine aminotransferase (ALT), liver steatosis, and increased proinflammatory cytokines in the liver. TREM-1 messenger RNA (mRNA) expression was significantly increased in alcohol-fed mice, and TREM-1 inhibitors significantly reduced this increase. TREM-1 inhibition significantly attenuated alcohol-induced spleen tyrosine kinase (SYK) activation, an early event in both TLR4 and TREM-1 signaling. The TREM-1 inhibitors significantly inhibited macrophage (epidermal growth factor-like module-containing mucin-like hormone receptor-like 1 [F4/80], clusters of differentiation [CD]68) and neutrophil (lymphocyte antigen 6 complex, locus G [Ly6G] and myeloperoxidase [MPO]) markers and proinflammatory cytokines (monocyte chemoattractant protein 1 [MCP-1], tumor necrosis factor α [TNF-α], interleukin-1β [IL-1β], macrophage inflammatory protein 1α [MIP-1α]) at the mRNA level compared to the HDL vehicle. Administration of TREM-1 inhibitors ameliorated liver steatosis and early fibrosis markers (α-smooth muscle actin [αSMA] and procollagen1α [Pro-Col1α]) at the mRNA level in alcohol-fed mice. However, the HDL vehicle also reduced serum ALT and some cytokine protein levels in alcohol-fed mice, indicating HDL-related effects. Conclusion: HDL-delivered novel TREM-1 peptide inhibitors ameliorate early phases of inflammation and neutrophil and macrophage recruitment and activation in the liver and attenuate hepatocyte damage and liver steatosis. TREM-1 inhibition represents a promising therapeutic approach for further investigations in ALD.

Figure 1

Impact of TREM‐1 pathway inhibition on TREM‐1 and inflammatory cytokine mRNA expression. [Correction made 31 October, 2018. The table title was modified to the correct description.] TREM‐1 pathway inhibition suppresses the expression of (A) TREM‐1 and inflammatory cytokines (B) MCP‐1, (C) TNF‐α, (D) IL‐1β, and (E) MIP‐1α but not (F) RANTES at the mRNA level as measured in whole‐liver lysates by real‐time quantitative PCR. * indicates significance level compared to nontreated PF group; ^# indicates significance level compared to nontreated alcohol‐fed group; ^o indicates significance level compared to vehicle‐treated alcohol‐fed group. Significance levels are as follows: *^/#/o P ≤ 0.05; **^/##/oo P ≤ 0.01; ***^/ooo P ≤ 0.001; ****P ≤ 0.0001. Abbreviation: CCL, chemokine (C‐C motif) ligand.

Figure 2

TREM‐1 blockade and protein levels of inflammatory pathway molecules. [Correction made 31 October, 2018. The table title was modified to the correct description.] TREM‐1 blockade reduces inflammatory cytokine levels in (A) serum and (B‐D) whole‐liver lysates as measured with specific ELISA kits. (E‐G) Total liver protein was analyzed for total SYK and activated p‐SYK^Y525/526 expression by western blotting using β‐actin as a loading control. Statistical analysis was performed by evaluating two blots (n = 4/group).* indicates significance level compared to the nontreated PF group; ^# indicates significance level compared to the nontreated alcohol‐fed group; ^o indicates significance level compared to the vehicle‐treated alcohol‐fed group. Significance levels are as follows: *^/#/o P ≤ 0.05; **^/## P ≤ 0.01; ***P ≤ 0.001; ****^/#### P ≤ 0.0001.

Figure 3

TREM‐1 inhibition and markers of immune cell infiltration. [Correction made 31 October, 2018. The table title was modified to the correct description.] (A,B) TREM‐1 inhibition suppresses the mRNA expression of macrophage cell markers in the liver as measured by real‐time quantitative PCR. (C,D) Both TREM‐1 inhibitors attenuated F4/80 as shown by IHC. (E,F) TREM‐1 inhibition suppresses the mRNA expression of neutrophil cell markers in the liver as measured by real‐time quantitative PCR. (G,H) Both TREM‐1 inhibitors attenuated MPO‐positive cell infiltration as shown by IHC. * indicates significance level compared to the nontreated PF group; ^# indicates significance level compared to the nontreated alcohol‐fed group; ^o indicates significance level compared to the vehicle‐treated alcohol‐fed group. Significance levels are as follows: *^/#/o P ≤ 0.05; **^/## P ≤ 0.01; ^### P ≤ 0.001; ****^/#### P ≤ 0.0001.

Figure 4

Liver damage after 5 weeks of alcohol feeding and effect of TREM‐1 pathway inhibition in a mouse model of ALD. Cheek blood and livers were harvested at death. (A) Serum ALT levels were measured using a kinetic method. (B‐D) Liver sections were stained with (B,C) Oil Red O and (D) H&E staining, and the lipid content was analyzed by ImageJ (B). * indicates significance level compared to the nontreated PF group; ^# indicates significance level compared to the nontreated alcohol‐fed group; ^o indicates significance level compared to the vehicle‐treated alcohol‐fed group. Significance levels are as follows: *^/o P ≤ 0.05; ^##/oo P ≤ 0.01;***^/### P ≤ 0.001; ****P ≤ 0.0001.

Figure 5

TREM‐1 inhibition alters mRNA expression of enzymes involved in lipid metabolism. mRNA expression of genes involved in (A,B) lipid synthesis (SERBF1, ACC1), (D‐F) lipid oxidation (PPARα, CPT1A, MCAD), and (C) the lipid accumulation marker (ADRP) were measured in whole liver. [Correction made 31 October, 2018. The table title and label descriptions included the wrong information and have been modified.] * indicates significance level compared to the nontreated PF group; ^# indicates significance level compared to the nontreated alcohol‐fed group; ^o indicates significance level compared to the vehicle‐treated alcohol‐fed group. Significance levels are as follows: *^/#/o P ≤ 0.05; **^/##/oo P ≤ 0.01; ^### P ≤ 0.001; ****P ≤ 0.0001.

Figure 6

TREM‐1 inhibition suppresses the expression of fibrinogenesis marker molecules. (A) Pro‐Col1α and (B) α‐SMA are suppressed by TREM‐1 blockade at the RNA level as measured in whole‐liver lysates. [Correction made 31 October, 2018. The label descriptions included the wrong description and have been modified.] * indicates significance level compared to the nontreated PF group; ^# indicates significance level compared to the nontreated alcohol‐fed group; ^o indicates significance level compared to the vehicle‐treated alcohol‐fed group. Significance levels are as follows: *^/#/o P ≤ 0.05; ^## P ≤ 0.01; ****^/#### P ≤ 0.0001. Data are presented as mean ± SEM.

Figure 7

Scavenger receptors SR‐A and SR‐BI mediate the macrophage uptake of GF9‐HDL and GA/E31‐HDL. (A) Schematic representation for the proposed role of TREM‐1 signaling in ALD and the SCHOOL mechanism of TREM‐1 blockade. (A, left panel) Activation of the TREM‐1/DAP12 receptor complex expressed on Kupffer cells leads to phosphorylation of the DAP12 cytoplasmic signaling domain, subsequent SYK recruitment, and the downstream inflammatory cytokine response. (A, right panel) SR‐mediated endocytosis of HDL‐bound GF9 peptide inhibitors by Kupffer cells results in the release of GF9 (GA31 or GE31) into the cytoplasm; GF9 self‐penetrates the cell membrane and blocks intramembrane interactions between TREM‐1 and DAP12, thereby preventing DAP12 phosphorylation and the downstream signaling cascade. (B,C) Macrophage uptake of GF9‐HDL and GA/E31‐HDL in vitro is SR mediated in a time‐dependent manner and is largely driven by SR‐A. As described in the Materials and Methods, J774 macrophages were cultured at 37°C overnight with medium. Prior to uptake of GF9‐HDL and GA/E31‐HDL, cells were treated for 1 hour at 37°C with 40 μM cytochalasin D and either (B) 400 μg/mL fucoidan or (C) 10 μM BLT‐1, as indicated. Cells were then incubated for either 4 hours or 22 hours with medium containing 2 μM rho B‐labeled GF9‐HDL (gray bars) or GA/E31‐HDL (black bars), respectively. Cells were lysed, and rho B fluorescence intensities of lysates were measured and normalized to the protein content. Results are expressed as mean ± SEM (n = 3); *P ≤ 0.05; **P ≤ 0.01; ****P ≤ 0.0001 versus uptake of GF9‐HDL and GA/E31‐HDL in the absence of inhibitor. Abbreviations: D, DAP12; DAP12, DNAX activation protein of 12 kDa; K, Kupffer cell; RFU, relative fluorescence units; SCHOOL, signaling chain homo‐oligomerization.