R.J. Hollis1,2,3, M. Aziz1,2,3, G. Ma1, P. Wang1,2,3 1The Feinstein Institutes For Medical Research, Center For Immunology And Inflammation, Manhasset, NY, USA 2Zucker School Of Medicine At Hofstra/Northwell Health, Department Of Surgery, Manhasset, NY, USA 3Elmezzi Graduate School Of Molecular Medicine, Manhasset, NY, USA
Introduction:
Mesenteric ischemia results from loss of perfusion to the small intestines due to obstruction of the superior mesenteric artery (SMA). Restoration of perfusion causes inflammation via circulation of byproducts from cell death, such as extracellular cold-inducible RNA-binding protein (eCIRP), a damage-associated molecular pattern (DAMP) that dysregulates immune function in mesenteric ischemia/reperfusion (I/R). This process leads to organ damage, like acute respiratory distress syndrome, and sepsis from disruption of the intestinal barrier. Apoptosis inhibitor in macrophages (AIM) is a multifunctional protein secreted by macrophages. AIM has been studied in mouse models of sepsis, but its role in mesenteric I/R is unknown. We hypothesized that AIM expression is decreased in mesenteric I/R and that AIM treatment reduces eCIRP-induced inflammation by restoring normal metabolic function.
Methods:
Mice underwent laparotomy with occlusion of the SMA for 60 min, followed by reperfusion for 4 h before blood and tissue collection. Expression levels of AIM mRNA in the gut and lungs were evaluated with qPCR, and protein levels were assessed with western blot. RAW 264.7 macrophages were pretreated with recombinant mouse AIM (rmAIM) and stimulated with eCIRP. The supernatant was collected for ELISA, and RNA was extracted from cells for qPCR. Mitochondrial respiration, measured as oxygen consumption rate (OCR), and ATP levels were detected using Seahorse metabolic assays.
Results:
Mesenteric I/R resulted in a marked decrease in the expression of AIM in gut tissues, with mRNA and protein levels reduced by 46% and 44%, respectively, compared to sham. The expression of AIM mRNA and protein in the lungs decreased by 25% and 27%, respectively. Mesenteric I/R increased serum levels of eCIRP. Treatment of macrophages with eCIRP (1 µg/mL) significantly increased the mRNA expression of TNFα and IL-1β by 30.5- and 271-fold, respectively. Interestingly, pretreatment of eCIRP-stimulated macrophages with rmAIM (2 µg/mL) decreased the mRNA expression of TNFα and IL-1β by 27% and 39%, respectively. Additionally, we found decreased protein expression of IL-6 and TNFα by 39% and 18%, respectively, in rmAIM-treated macrophages after eCIRP stimulation. Lastly, rmAIM recovered mitochondrial function, marked by 19% and 25% improved OCR and ATP levels, respectively (Table 1).
Conclusion:
Our data demonstrate that AIM levels are decreased in the intestine and lungs after mesenteric I/R and rmAIM reduces proinflammatory cytokines, highlighting the anti-inflammatory role of AIM. It is expected that supplementation of this protein has potential to treat patients with mesenteric I/R injury.