M. Hough1,2, M. O’Guinn1,2, D. Handler1,2, E. Lim1,2, C.P. Gayer1,2,3 1Children’s Hospital Los Angeles, The Saban Research Institute, Los Angeles, CA, USA 2Children’s Hospital Los Angeles, Division Of Pediatric Surgery, Los Angeles, CA, USA 3University Of Southern California, Division Of Pediatric Surgery, Los Angeles, CA, USA
Introduction:
Farnesoid-X receptor (FXR) is a nuclear bile acid receptor known to regulate the inflammatory response. We have previously shown that the intestinal barrier of FXR knockout (FXRKO) animals is preserved in acute injury models, including lipopolysaccharide (LPS) injection, dithizone/Klebsiella, and cecal ligation/puncture, and is associated with reduced inflammatory cytokine production and preserved tight junction architecture. Macrophages express FXR, and are essential in inflammatory immune response. We hypothesize that macrophages-specific FXRKO (mpFXRKO) will have altered cytokine expression, including increased I1b (pro-inflammatory) and decreased Il10 (anti-inflammatory) expression. We also hypothesize mpFXRKO mice as compared to wild type (WT) mice would have an attenuated cytokine response after LPS injury.
Methods:
Bone marrow from WT and mpFXRKO mice was harvested and cultured in RPMI media with m-CSF (macrophage colony-stimulating factor). Bone marrow derived macrophages (BMDM) were then maintained in naïve state or polarized to M1 (pro-inflammatory phenotype) or M2 (anti-inflammatory phenotype). I1b and Il10 cytokine expression was measured with qPCR six hours after polarization. Additionally, WT and mpFXRKO mice were injected with LPS or normal saline for control. Terminal ileum was harvested and mucosal scrapings collected. qPCR then evaluated expression of Il1a, I1b, Tnf, Il6, Il10 and Cxcl1 at 16 hours and two hours.
Results:
Compared to WT macrophages, naïve mpFXRKO macrophages had an increased expression of I1b, M1 mpFXRKO macrophages had a decreased expression of I1b, and unexpectedly, M2 mpFXRKO macrophages had an increased expression of I1b. There was no significant change in Il10 expression across all three phenotypes between WT and mpFXRKO macrophages. When comparing cytokine expression at 16 and two hours, there was an increase in Il6 expression in mpFXRKO compared to WT mice, with no significant change in Il1a, Il1b, Tnf, Il10, and Cxcl1.
Conclusion:
These data indicate FXR attenuates the inflammatory response in macrophages. Its absence in the M0 naïve phenotype potentially leads to a baseline inflammatory state, increasing cytokine Il1b, however then the decreased expression in the M1 proinflammatory phenotype suggests a possible protective aspect in mpFXRKO animals that are in an injured state. These findings warrant further investigation to delineate immunomodulatory effects of the absence of FXR on macrophages, and their ultimate influence on intestinal injury.