Limited macrophage dynamics in progressing or regressing murine atherosclerotic plaques

Previous studies using particle labeled monocytes to assess recruitment into tissues have demonstrated that bead labeling did not affect the ability of monocytes to enter tissues (Tacke et al., 2007), and particle uptake did not activate p38/MAPK or NFκB pathways (Yates and Russell, 2005). However, the possibility remains that bead labeling could have effects on global gene expression profiles. To address this prospect, we performed gene expression profiling of classical and non-classical monocytes that had taken up latex particle in vivo against cells from the same mouse that had not taken up particles. 8 samples were analyzed with two biological replicates per condition: Gr1 high monocytes with or without beads and Gr1 low monocytes with or without beads. C57bl/6 mice were injected i.v. with latex beads (diluted 1:4 in PBS) three days following chodonate liposome mediate monocyte depletion, then four days later classical and nonclassical blood monocytes were FACS sorted (FACSAria, BD Biosciences) for presence or absence of latex beads. RNA was extracted from sorted cells by TRIzol and RNeasy mRNA isolation kit (Qiagen). RNA amplification, direct labeling, hybridization, and chip scanning were performed as previously described (Grabner et al., 2009). Microarray was performed on an Affymetrix GeneChip 430 2.0. R packages 'oligo' and 'limma' were used to read, normalize, access QC and perform DE from the microarray expression data. Gene set enrichment analysis with fgsea package was used to identify pathway enrichments in hallmark and canonical pathways. Benjamini-Hochberg correction was used to correct p-value for multiple comparisons.