5,5-Dimethyl-1-pyrroline N-oxide Modulates Transcriptome and Interactome Towards Dampening Innate Immune Response in RAW 264.7 cells

The nitrone spin trap 5,5-dimethyl-1-pyrroline N -oxide (DMPO) was originally synthesized for the study of free radicals. However we found that DMPO can also prevent inflammatory activation in RAW264.7 cells primed with lipopolysaccharide (LPS). Additionally, DMPO prevents lung damage, adipose tissue inflammation, systemic inflammation and insulin resistance in a mouse model of acute distress respiratory syndrome induced by intratracheal instillation with LPS. These effects of DMPO are more likely caused by changes in gene expression in innate immune cells. Thus herein we tested whether DMPO by itself can change the transcriptome and interactome in RAW 264.7 cells, as a model of macrophage. To accomplish our goal we incubated RAW 264.7 cells with (DMPO) or without (control) 50 mM DMPO for 6h. The transcriptome was analyzed using micro-array (Illumina) and corroborated by Nanostring (nCounter) technology. Bioinformatics analysis showed 79 differentially expressed genes (DEGs) in DMPO vs control comparison (ONE-way ANOVA; FDR = 0.05). DAVID databases for identifying enriched Gene Onthology terms and Ingenuity Pathway Analysis (IPA) for functional analysis showed that DMPO DEGs were consistent with a negative regulation of innate immune response. Functional analysis indicated that IRF7 and TLRs were related (predicted inhibitions) to the observed transcriptomic effects of DMPO. Functional data analyses are consistent with DMPO dampening LPS-induced inflammatory activation of RAW 264.7 cells by down regulating several genes related to the innate immune response. These effects were also supported by Nanostring data showing that DMPO downregulates the expression of inflammation markers (CCL2, IFNβ, COX-2 and iNOS). Remarkably, our data suggest that DMPO by itself shifts the transcriptomic profile of RAW264.7 cells towards a negative modulation of innate immune response.