Arf6 is a small GTPase regulating many cellular processes including cytoskeletal remodeling, receptor endocytosis, and phagocytosis of pathogens. Arf6 knockdown in neutrophil (PMN)-like cells was reported to inhibit chemotactic peptide-mediated activation of phospholipase D, the oxidative burst, and 2 integrin-dependent adhesion. In mice, the migration of PMNs knock out for Arf6 to the site of inflammation was diminished and associated with reduced cell surface expression of 2 integrins. Conditional knockout mice lacking Arf6 in PMNs were used to assess the impact of Arf6 depletion on the functions and gene expression profile of PMNs isolated from the mouse air pouch injected with lipopolysaccharide (LPS). The expression of several genes was modulated in PMNs-Arf6 cKO with Lpar6 and Lacc-1 being the most up-regulated and down-regulated genes, respectively. Decreased expressed of Lacc-1 was validated at the protein level in PMN-Arf6 cKO, and silencing of Arf6 in THP-1 monocytic cells delayed LPS-mediated Lacc-1 expression. Here we report that fMLP or zymosan-induced glycolysis and oxygen consumption rate were decreased in air pouch PMNs but not in BM PMNs of Arf6 cKO mice when compared to control floxed cells. Reduced oxygen consumption correlated with decreased production of superoxide and ROS. Depletion of Arf6 in mouse PMNs also reduced phagocytosis and interfered apoptosis. The data suggest that Arf6 regulates energy metabolism, which may contribute to impaired phagocytosis, ROS production, and apoptosis in PMN-Arf6 cKO. This study provides new information on the functions and the inflammatory pathways influenced by Arf6 in PMNs.
Overall design: Leucocytes that have migrated into the mouse air pouches injected with LPS were collected from the Arf6 cKO and the control groups (homozygous Cre+/+ and flox+/+Cre-/- mice, respectively). For each mouse genotype, three independent groups of 5 mice were used. Air pouch PMNs were pooled and purified by negative selection as described above. The cell suspensions were 98% Ly6G positive as estimated by flow cytometry (Supplemental Fig. 1). Mouse PMNs were lysed in TRIzol reagent for total mRNA extraction. RNA extraction and quality check of all RNA samples before the Affymetrix’s GeneChip arrays were performed at the CRCHU of Québec-Laval University Genomics Platform (CHU de Québec-Université Laval Research Center, Québec, QC, Canada). Briefly, 100 ng of total RNA was labeled using FlashTag Biotin HSR (Genisphere, Hatfield, PA) and hybridized on mouse Gene 2.0 ST-Pico Kit (Affymetrix, Santa Clara, CA). Total RNA was used as the input fraction. Arrays were scanned on a GeneChip scanner 3000 G7 (Affymetrix, Santa Clara, CA), . The image data were analyzed by using the Affymetrix Expression Console Software to perform the quality control, the background subtraction and the normalization of probe set intensities with the method of Robust Multiarray Analysis (RMA). Microarray analyses were performed by the CHU de Québec Research Center (CHUL) Gene Expression Platform, Quebec, Canada.
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