The proinflammatory response leads to various types of pathologic pathways, including the development of preterm birth. Preterm birth occurs in 12% of deliveries in the United States and causes more than 70% of perinatal morbidity and mortality. The most common cause of spontaneous preterm birth is intrauterine infection in the mother. There is accumulating evidence indicating that the release of proinflammatory cytokines plays a critical role in the pathogenesis of inflammation-associated premature delivery. We found that the common organic solvent, N,N-dimethylacetamide (DMA), prevents endotoxin-induced preterm birth in timed pregnant C57BL/6 embryonic day (E)15.5 mice and rescues their pups from spontaneous abortion at doses many-fold lower than those currently used clinically and in a dose-dependent fashion. We also provide histologic evidence that DMA suppresses the endotoxin-triggered proinflammatory response by significantly attenuating inflammatory cell infiltration of placental tissue. Furthermore, immunoblotting analysis of placental tissue harvested from our murine models revealed DMA-mediated regulation of expression of the proinflammatory cytokines IL-1β, tumor necrosis factor α, and IL-6, and increased expression of the regulatory inflammatory cytokine IL-10. By using in vitro studies, we provide evidence that DMA suppresses macrophage function and that this small molecule prevents nuclear translocation of nuclear factor-kB. These results suggest that DMA represents a newly discovered, nontoxic therapy for a broad range of inflammatory disorders.

PW is an inventor of patent applications (WO/2010/120726 and 61/881.798) covering the fundamental concept of targeting cold-inducible RNAbinding protein for the treatment of inflammatory diseases, licensed by TheraSource LLC. PW and MZ are cofounders of TheraSource LLC.

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.

In sepsis, macrophage bacterial phagocytosis is impaired, but the mechanism is not well elucidated. Extracellular cold-inducible RNA-binding protein (eCIRP) is a damage-associated molecular pattern that causes inflammation. However, whether eCIRP regulates macrophage bacterial phagocytosis is unknown. Here, we reported that the bacterial loads in the blood and peritoneal fluid were decreased in CIRP−/− mice and anti-eCIRP Ab-treated mice after sepsis. Increased eCIRP levels were correlated with decreased bacterial clearance in septic mice. CIRP−/− mice showed a marked increase in survival after sepsis. Recombinant murine CIRP (rmCIRP) significantly decreased the phagocytosis of bacteria by macrophages in vivo and in vitro. rmCIRP decreased the protein expression of actin-binding proteins, ARP2, and p-cofilin in macrophages. rmCIRP significantly downregulated the protein expression of βPIX, a Rac1 activator. We further demonstrated that STAT3 and βPIX formed a complex following rmCIRP treatment, preventing βPIX from activating Rac1. We also found that eCIRP-induced STAT3 phosphorylation was required for eCIRP’s action in actin remodeling. Inhibition of STAT3 phosphorylation prevented the formation of the STAT3-βPIX complex, restoring ARP2 and p-cofilin expression and membrane protrusion in rmCIRP-treated macrophages. The STAT3 inhibitor stattic rescued the macrophage phagocytic dysfunction induced by rmCIRP. Thus, we identified a novel mechanism of macrophage phagocytic dysfunction caused by eCIRP, which provides a new therapeutic target to ameliorate sepsis.

Premature delivery occurs in 12% of all births, accounts for nearly half of neonatal morbidity and is increasing in frequency. Current therapeutic approaches to preterm delivery are ineffective and present serious risks to both the mother and fetus. Although there are multiple factors that contribute to the etiology of preterm birth, the single most common cause is infection. Recently, using cDNA microarray analysis of human placental tissue, we demonstrated that human placental matrix metalloproteinase-1 (MMP-1) is upregulated during labor. In a separate line of investigation, we have shown that blockade of endothelin-1 (ET-1) action through the use of an endothelin-converting enzyme-1 (ECE-1) inhibitor, an established commercially available endothelin receptor antagonist or a novel quinolone-derived endothelin receptor antagonist synthesized by our group also prevents preterm labor and delivery in a mouse model. We have now shown that induction of preterm labor with lipopolysaccharide in our mouse model is associated with increased levels of MMP-1. Furthermore, we showed that silencing the ECE-1/ET-1 pathway by using ECE-1 RNA interference prevents both the onset of preterm labor and upregulation of MMP-1. The data indicate that ET-1 and MMP-1 act in the same molecular pathway in preterm labor.