Sa1765 Defective Autophagy Contributes to Impaired Bacterial Clearance of SAMP1/YITFC Mice

Abstract

Background: The commensal microbiota are known to regulate the activity of neutrophils and other leukocytes in the intestine, but the impact of the microbiota on systemic neutrophil activity remains unknown. Serum amyloid A (Saa) is a circulating HDL-associated apolipoprotein and acute phase protein. Saa genes are expressed by multiple tissues and cell types and are markedly induced by diverse stimuli including the gut microbiota. However, the precise roles of Saa in inflammation remain elusive because both pro-and anti-inflammatory actions have been reported. Aim: Assess the effects of microbiota colonization and Saa induction on systemic neutrophil development and function. Methods: The effect of the microbiota on neutrophil localization and migration was evaluated using germ-free (GF) and conventionalized (CONVD) transgenic zebrafish expressing GFP in neutrophils (Tg(mpx:GFP)) Flow cytometry was used to quantify and sort GFP(+) neutrophils for qRTPCR ofmpxmRNA levels. Neutrophil migrationwas assessed using tracking analysis of GFP(+) neutrophils using Volocity. Neutrophil recruitment was assessed using a tail-wounding assay. Morpholinos targeting saa were used on GF and CONVD fish to evaluate the role of saa on neutrophil biology. In vitro studies were performed using PAC-2 zebrafish embryonic fibroblast cells and peritoneal isolated murine neutrophils. NF-κB pathway activation was measured in SAA stimulated PAC-2 cells by western blot and through transfection of a pikbaa:Luc reporter. Murine neutrophil migration in response to SAA in the presence of an NF-κB inhibitor was assessed using a transwell system. Results: The presence of a microbiota resulted in increased neutrophil number (~3 fold, p<0.05) and myeloperoxidase expression (~5 fold, p<0.05)), and altered neutrophil localization and migratory behaviors (~2 fold increased velocity and increased directional migration). These effects of the microbiota on neutrophil homeostasis were accompanied by an increased recruitment of neutrophils to the injury site. Morpholino knockdown experiments revealed saamediates microbial stimulation of tissue-specific neutrophil migratory behaviors. In vitro studies revealed that zebrafish cells respond to Saa exposure by activating NF-κB (~3 fold, p<0.001), and that Saa-dependent neutrophil migration requires NF-κB-dependent gene expression.Conclusions:These results implicate the commensal microbiota as an important environmental factor regulating diverse aspects of systemic neutrophil development and function, and reveal a critical role for a Saa-NF-κB signaling axis in mediating neutrophil migratory responses.