Inhibition of the Interaction of TREM-1 and eCIRP Attenuates Inflammation and Improves Survival in Hepatic Ischemia/Reperfusion.

INTRODUCTION: Triggering receptor expressed on myeloid cells-1 (TREM-1) has important implications in sepsis and inflammation and is a novel receptor for extracellular cold-inducible RNA-binding protein (eCIRP). We hypothesize that the inhibition of TREM-1 via its interaction with eCIRP by novel peptide inhibitor M3 or knockout gene will attenuate the inflammation and injury associated with severe hepatic ischemia/reperfusion (I/R). METHODS: Wild-type (WT) C57BL/6 and TREM-1-/- mice underwent 60 min of 70% hepatic ischemia, with 24 h of reperfusion. Additionally, WT mice underwent hepatic I/R and were treated with M3 (10 mg/kg body weight) or vehicle (normal saline) at the start of reperfusion. Blood and ischemic liver tissues were collected, and analysis was performed using enzymatic assays, enzyme-linked immunosorbent assay, reverse-transcription quantitative polymerase chain reaction, and pathohistology techniques. For survival surgery, mice additionally underwent resection of non-ischemic lobes of the liver and survival was monitored for 10 days. RESULTS: There was an increase in serum levels of tissue markers including aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase as well as cytokine levels (IL-6) and histological scoring of hematoxylin and eosin sections in WT I/R mice. These markers decreased substantially in TREM-1-/- mice. Additionally, neutrophil infiltration markers and markers of local inflammation (myeloperoxidase, macrophage inflammatory protein-2, cyclooxygenase-2) were attenuated in TREM-1-/- mice. Similarly, we show a significant decrease in injury and inflammation markers with M3 treatment. Additionally, we demonstrate decreased apoptosis with TREM-1 inhibition. Finally, M3 treatment improved the survival rate from 42% to 75% after hepatic I/R. CONCLUSION: TREM-1 is an important eCIRP receptor in the inflammatory response of hepatic I/R, and deficiency of TREM-1 via knockout gene or peptide inhibition attenuated liver injury and inflammation, and improved survival. Inhibition of the TREM-1 and eCIRP interaction in hepatic I/R may have important therapeutic potential.

TREM-1 amplifies trophoblastic inflammation via activating NF-κB pathway during preeclampsia.

INTRODUCTION: Excessive activation of maternal systemic inflammation is one of the underlying causes of pathology during the disease course of preeclampsia (PE). The triggering receptor expressed on myeloid cells-1 (TREM-1) participates in the development and persistence of inflammation. We hypothesized that dysregulated TREM-1 may be involved in the pathogenesis of PE by promoting the secretion of trophoblastic pro-inflammatory cytokines that augment inflammation. METHODS: The localization of TREM-1 in placenta and the extravillous trophoblast cell line (TEV-1) was determined by immunohistochemical staining. The expression level of TREM-1 and pro-inflammatory cytokines in placentas were compared between normal pregnancies and PE. We used lipopolysaccharide (LPS) to simulate trophoblastic inflammation. TEV-1 cells were transfected with TREM-1 plasmid and si-TREM-1 respectively, and then were incubated with LPS. The expression levels of pro-inflammatory cytokines and key molecules featured in nuclear transcription factor-kappaB (NF-κB) pathway were detected. Transwell assays were used to detect the effects of TREM-1 on cell migration and invasion. RESULTS: TREM-1 was localized on both villous trophoblasts (VTs) and extravillous trophoblasts (EVTs). TREM-1 and pro-inflammatory cytokines were up-regulated in preeclamptic placenta. Overexpression of TREM-1 promoted the activation of NF-κB pathway and the release of pro-inflammatory factors induced by LPS, and enhanced migration and invasion of TEV-1 cells. Inhibition of TREM-1 significantly attenuated LPS-induced effects and suppressed migration and invasion. DISCUSSION: This study suggested that TREM-1 was up-regulated in PE, and may promote the production of downstream inflammatory factors by activating NF-κB pathway in trophoblastic cells, thus exerting pro-inflammatory effects in the pathogenesis of PE.

LR12 Promotes Liver Repair by Improving the Resolution of Inflammation and Liver Regeneration in Mice with Thioacetamide- (TAA-) Induced Acute Liver Failure.

BACKGROUND: Triggering receptor expressed on myeloid cells-1 (TREM-1) controls the mobilization of inflammatory cells in response to injury and consequently enhances liver damage. LR12 is a TREM-1 inhibitory peptide. However, the role of LR12 in acute liver failure (ALF) has remained elusive. This study was aimed at indicating whether LR12 could promote liver repair in mice with thioacetamide- (TAA-) induced ALF. METHODS: BALB/c mice were intraperitoneally injected with TAA, followed by intravenous injection of LR12. Damage and regeneration of the liver were assessed. LO2 cells and macrophages were used to assess the therapeutic effects of LR12. RESULTS: Mice treated with TAA for 24 h developed ALF, while liver inflammation was alleviated after LR12 treatment. Moreover, LR12 promoted hepatocyte regeneration in mice with TAA-induced ALF. In vitro, the supernatant from TAA+LR12-treated macrophages promoted the proliferation of LO2 cells. Cytokine protein microarray analysis suggested that LR12 promoted the secretion of C-C chemokine ligand 20 (CCL20) from macrophages. Besides, neutralization of CCL20 blocked the effects of LR12, thus inhibited the proliferation of LO2 cells in vitro, aggregated the liver inflammation, and restrained hepatocyte regeneration in ALF mice in vivo. Furthermore, we also found that LR12 activated the p38 mitogen-activated protein kinase (MAPK) pathway in hepatocytes through promoting the secretion of CCL20 from macrophages. CONCLUSIONS: LR12 could improve the resolution of inflammation and liver regeneration in mice with TAA-induced ALF by promoting the secretion of CCL20 from macrophages and activating the p38 MAPK pathway. Therefore, LR12 could be an attractive therapeutic target for the treatment of ALF.

Extracellular CIRP and TREM-1 axis promotes ICAM-1-Rho-mediated NETosis in sepsis.

Extracellular cold-inducible RNA-binding protein (eCIRP) is a damage-associated molecular pattern (DAMP). Intercellular adhesion molecule-1 (ICAM-1) expressing neutrophils produce excessive amounts of neutrophil extracellular traps (NETs). We reveal that eCIRP generates ICAM-1+ neutrophils through triggering receptor expressed on myeloid cells-1 (TREM-1) and the ICAM-1+ neutrophils involve Rho GTPase to promote NETosis. Treatment of BMDN with rmCIRP increased the frequency of ICAM-1+ BMDN, while rmCIRP-treated TREM-1-/- BMDN or pretreatment of BMDN with TREM-1 inhibitor LP17 significantly decreased the frequency of ICAM-1+ neutrophils. The frequencies of ICAM-1+ neutrophils in blood and lungs were markedly decreased in rmCIRP-injected mice or septic mice treated with LP17. Coculture of ICAM-1-/- neutrophils or wild-type (WT) neutrophils with WT macrophages in the presence of a peptidylarginine deiminase 4 (PAD4) inhibitor reduced TNF-α and IL-6 compared to WT neutrophils treated with rmCIRP. Treatment of ICAM-1-/- neutrophils with rmCIRP resulted in reduced quantities of NETs compared to WT rmCIRP-treated neutrophils. Treatment of BMDN with rmCIRP-induced Rho activation, while blockade of ICAM-1 significantly decreased Rho activation. Inhibition of Rho significantly decreased rmCIRP-induced NET formation in BMDN. TREM-1 plays a critical role in the eCIRP-mediated increase of ICAM-1 expression in neutrophils, leading to the increased NET formation via Rho activation to exaggerate inflammation.

Research on mechanism of PCS in damaging vascular endothelial cells and promoting formation of atherosclerosis via TLR4/TREM-1.

OBJECTIVE: The study aimed to explore the effects of p-cresyl sulfate (PCS) of damaging vascular endothelial cells and promoting the formation of atherosclerosis in mice. MATERIALS AND METHODS: The apolipoprotein E (ApoE)-/- mice were fed normally and with a high-fat diet; the ApoE-/- mice fed with high-fat diet were divided into two groups and treated with blank control and PCS, respectively. The aortic arch in each group was taken and underwent the oil red O staining, and the serum PCS content in each group was detected. The basic components of plaque were observed, including foam cells, lipid deposition, and cholesterol crystal. Moreover, human umbilical vein endothelial cells were cultured and divided into control group, PCS treatment group (PCS), PCS treatment with TLR4 overexpression group (PCS+TLR4+), and PCS treatment with TLR4 knock-out group (PCS+TLR4-). The degree of endothelial cell damage was detected using a cluster of differentiation CD42b-/CD31+ endothelial microparticles (EMPs), and expressions of Toll-like receptor 4 (TLR4), triggering receptor expressed on myeloid cells-1 (TREM-1), phosphorylated-endothelial nitric oxide synthase (p-eNOS), and tumor necrosis factor-α (TNF-α) in cells were detected via Polymerase Chain Reaction (PCR) and Western blotting. RESULTS: The serum PCS concentration in high-fat ApoE-/- mice was increased, and the aortic arch sections of ApoE-/- mice treated with PCS displayed the evident atherosclerotic plaques. Experimental results of human umbilical vein endothelial cells showed that the activity of human umbilical vein endothelial cells treated with PCS declined, the expression levels of TLR4, TREM-1, and TNF-α were increased, while that of p-eNOS was decreased. After the TLR4 knockout, the above effects of PCS were reversed. CONCLUSIONS: PCS damages vascular endothelial cells through TRL4/TREM-1, thereby accelerating the formation of atherosclerosis.

Suppression of inflammatory and infection responses in lung macrophages by eucalyptus oil and its constituent 1,8-cineole: Role of pattern recognition receptors TREM-1 and NLRP3, the MAP kinase regulator MKP-1, and NFκB.

Eucalyptus oil (EO) used in traditional medicine continues to prove useful for aroma therapy in respiratory ailments; however, there is a paucity of information on its mechanism of action and active components. In this direction, we investigated EO and its dominant constituent 1,8-cineole (eucalyptol) using the murine lung alveolar macrophage (AM) cell line MH-S. In an LPS-induced AM inflammation model, pre-treatment with EO significantly reduced (P ≤0.01or 0.05) the pro-inflammatory mediators TNF-α, IL-1 (α and ß), and NO, albeit at a variable rate and extent; 1,8-cineole diminished IL-1 and IL-6. In a mycobacterial-infection AM model, EO pre-treatment or post-treatment significantly enhanced (P ≤0.01) the phagocytic activity and pathogen clearance. 1,8-cineole also significantly enhanced the pathogen clearance though the phagocytic activity was not significantly altered. EO or 1,8-cineole pre-treatment attenuated LPS-induced inflammatory signaling pathways at various levels accompanied by diminished inflammatory response. Among the pattern recognition receptors (PRRs) involved in LPS signaling, the TREM pathway surface receptor (TREM-1) was significantly downregulated. Importantly, the pre-treatments significantly downregulated (P ≤0.01) the intracellular PRR receptor NLRP3 of the inflammasome, which is consistent with the decrease in IL-1ß secretion. Of the shared downstream signaling cascade for these PRR pathways, there was significant attenuation of phosphorylation of the transcription factor NF-κB and p38 (but increased phosphorylation of the other two MAP kinases, ERK1/2 and JNK1/2). 1,8-cineole showed a similar general trend except for an opposite effect on NF-κB and JNK1/2. In this context, either pre-treatment caused a significant downregulation of MKP-1 phosphatase, a negative regulator of MAPKs. Collectively, our results demonstrate that the anti-inflammatory activity of EO and 1,8-cineole is modulated via selective downregulation of the PRR pathways, including PRR receptors (TREM-1 and NLRP3) and common downstream signaling cascade partners (NF-κB, MAPKs, MKP-1). To our knowledge, this is the first report on the modulatory role of TREM-1 and NLRP3 inflammasome pathways and the MAPK negative regulator MKP-1 in context of the anti-inflammatory potential of EO and its constituent 1,8-cineole.

TREM-1 promotes intestinal tumorigenesis.

Triggering receptor expressed on myeloid cells-1 (TREM-1) is a potent amplifier of pro-inflammatory innate immune responses. Increasing evidence suggests a role for TREM-1 not only in acute pathogen-induced reactions but also in chronic and non-infectious inflammatory disorders, including various types of cancer. Here, we demonstrate that genetic deficiency in Trem1 protects from colorectal cancer. In particular, Trem1 -/- mice exhibited reduced tumor numbers and load in an experimental model of inflammation-driven tumorigenesis. Gene expression analysis of Trem1 -/- versus Trem1 +/+ tumor tissue demonstrated distinct immune signatures. Whereas Trem1 -/- tumors showed an increased abundance of transcripts linked to adaptive immunity, Trem1 +/+ tumors were characterized by overexpression of innate pro-inflammatory genes associated with tumorigenesis. Compared to adjacent tumor-free colonic mucosa, expression of Trem1 was increased in murine and human colorectal tumors. Unexpectedly, TREM-1 was not detected on tumor-associated Ly6C- MHC class II+ macrophages. In contrast, TREM-1 was highly expressed by tumor-infiltrating neutrophils which represented the predominant myeloid population in Trem1 +/+ but not in Trem1 -/- tumors. Collectively, our findings demonstrate a clear role of TREM-1 for intestinal tumorigenesis and indicate TREM-1-expressing neutrophils as critical players in colorectal tumor development.

Attenuated viral hepatitis in Trem1-/- mice is associated with reduced inflammatory activity of neutrophils.

TREM1 (Triggering Receptor Expressed on Myeloid Cells 1) is a pro-inflammatory receptor expressed by phagocytes, which can also be released as a soluble molecule (sTREM1). The roles of TREM1 and sTREM1 in liver infection and inflammation are not clear. Here we show that patients with hepatitis B virus (HBV) or hepatitis C virus (HCV) infection manifest elevated serum levels of sTREM1. In mice, experimental viral hepatitis induced by infection with Lymphocytic Choriomeningitis Virus (LCMV)-WE was likewise associated with increased sTREM1 in serum and urine, and with increased TREM1 and its associated adapter molecule DAP12 in the liver. Trem1-/- mice showed accelerated clearance of LCMV-WE and manifested attenuated liver inflammation and injury. TREM1 expression in the liver of wild-type mice was mostly confined to infiltrating neutrophils, which responded to LCMV by secretion of CCL2 and TNF-α, and release of sTREM1. Accordingly, the production of CCL2 and TNF-α was decreased in the livers of LCMV-infected Trem1-/- mice, as compared to LCMV-infected wildtype mice. These findings indicate that TREM1 plays a role in viral hepatitis, in which it seems to aggravate the immunopathology associated with viral clearance, mainly by increasing the inflammatory activity of neutrophils.