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, enzymelinked 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.

Background Sepsis is caused by the dysregulated immune response due to an initial infection and results in significant morbidity and mortality in humans. Extracellular cold inducible RNA binding protein (eCIRP) is a novel mediator identified in sepsis. We have previously discovered that microRNA 130b-3p inhibits eCIRP mediated inflammation. As RNA mimics are very unstable in vivo, we hypothesize that an engineered miRNA 130b-3p mimic named PS-OMe miR130, improves stability of the miRNA by protection from nuclease activity. We further hypothesize that PS-OMe miR130 reduces not only eCIRP-mediated inflammation and but also acute lung injury in a murine model of polymicrobial sepsis. Methods Single stranded PS-OMe miR130 was synthesized and the binding affinity to eCIRP was evaluated using surface plasmon resonance (SPR) and computational modeling. Macrophages were treated with PS-OMe miR130 with and without eCIRP and cell supernatant analyzed for cytokines. In vitro stability and the in vivo half-life of PS-OMe miR130 were also assessed. The effect of PS-Ome miR130 on eCIRP’s binding to TLR4 was evaluated by SPR analysis and modeling. Finally, the effect of PS-OMe miR130 on inflammation and injury was assessed in a murine model of sepsis. Results We demonstrate via SPR and computational modeling that PS-OMe miR130 has a strong binding affinity to eCIRP. This engineered miRNA decreases eCIRP induced TNF-α and IL-6 proteins, and it is highly stable in vitro and has a long in vivo half-life. We further demonstrate that PS-OMe miR130 blocks eCIRP binding to its receptor TLR4. Finally, we show that PS-OMe miR130 inhibits inflammation and lung injury, and improves survival in murine sepsis. Conclusion PS-OMe miR130 can be developed as a novel therapeutic by inhibiting eCIRP-mediated inflammation and acute lung injury in sepsis.

INTRODUCTION: Extracellular cold-inducible RNA-binding protein (eCIRP) is a novel mediator of inflammation and tissue injury. It has been shown that miRNA 130b-3p acts as an endogenous inhibitor of eCIRP. Because RNA mimics are unstable after in vivo administration, we have chemically engineered miRNA 130b-3p mimic (named PS-OMe miR130) to improve its stability by protection from nuclease activity. We hypothesize that PS-OMe miR130 reduces eCIRP-mediated injury and inflammation in a murine model of hepatic ischemia/ reperfusion (I/R), a model of sterile inflammation. METHODS:Adult male mice underwent 70% hepatic ischemia for 60 minutes and 24-hour reperfusion. At the start of reperfusion, mice were treated intravenously with vehicle (phosphate-buffered saline) or PS-OMe miR130. Blood and liver tissue were collected after 24 hours for biochemical analysis. Apoptosis in the liver tissue was determined by transferase dUTP nick-end labeling assay. RESULTS:After hepatic I/R, organ injury markers including aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase significantly decreased after PS-OMe miR130 treatment. Furthermore, histological analysis of liver sections demonstrated significantly less injury in PS-OMe miR130 treatment mice versus vehicle mice. In addition, tumor necrosis factor α mRNA, interleukin-1β mRNA, and neutrophil infiltration (myeloperoxidase activity and granulocyte receptor 1 immunohistochemistry) were significantly attenuated after PS-OMe miR130 treatment. Finally, apoptosis significantly decreased in liver tissue after treatment. CONCLUSION:PS-OMe miR130 decreases eCIRP-mediated injury and inflammation in a murine model of hepatic I/R.