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

Tornai, David; Furi, Istvan; Shen, Zu T.; Sigalov, Alexander B.; Coban, Sahin; Szabo, Gyongyi

Previous reports showed that TREM-1 activation leads to the expression and release of proinflammatory cytokines and chemokines through nuclear factor κB activation, which also regulates the expression of TREM-1, providing a positive feedback loop on the expression of the receptor. (4) Proinflammatory cytokine expression is increased in ALD (1) (2) (3) 23, 24) ; therefore, we hypothesized that TREM-1 signaling contributes to the amplification of proinflammatory pathways in ALD. To evaluate this hypothesis, first we tested whole-liver mRNAs of EtOH-fed and PF mice with or without treatment with two different TREM-1 inhibitory formulations and a vehicle control in a 5-week alcohol administration model of ALD in mice. (25) We found that mRNA levels of TREM-1 and MCP-1 were significantly increased in livers of alcohol-fed mice compared to PF controls (Fig. 1A,B) . In contrast, in mice treated with the TREM-1 inhibitors, both GF9-HDL and GA/E31-HDL inhibited alcohol-related changes in TREM-1; in addition, MCP-1 mRNA levels corresponded to those of the PF controls (Fig. 1A,B) . Although induction of TNF-α and IL-1ß in alcohol-fed mice did not reach statistical significance compared to PF controls, TREM-1 blockade by GF9-HDL resulted in a significant inhibition of TNF-α mRNA in the alcohol-fed mice compared to vehicle treatment (Fig. 1C) , while IL-1ß mRNA expression was also significantly attenuated by both the GF9-HDL and GA/E31-HDL formulations in the alcohol-fed as well as in the PF groups (Fig. 1D ). MIP-1α mRNA levels were increased in alcohol-fed mice, but TREM-1 blockade with GF9-HDL or GA/E31-HDL significantly attenuated this increase compared to the vehicle control (Fig. 1E) . Regulated on activation, normal T cell expressed, and secreted (RANTES) mRNA levels did not change regardless of alcohol feeding or TREM-1 treatment (Fig. 1F ). Next, we used specific ELISA kits to assess the protein levels of cytokines in the serum and in wholeliver lysates (Fig. 2) . We found a significant increase in MCP-1 level in the serum and liver and TNF-α in the liver of alcohol-fed mice compared to PF controls ( Fig. 2A-D) . All these alcohol-induced increases were prevented both in the serum and liver by administration of either TREM-1 inhibitor. Interestingly, we found attenuation of alcohol-induced liver MCP-1 and TNF-α induction even in the vehicle-treated (HDL only) groups ( Fig. 2A-C) . The increase in total IL-1ß levels after alcohol feeding and its attenuation by TREM-1 inhibition did not reach statistical significance (Fig. 2D) .