Macrophages are crucial for the initiation and resolution of an inflammatory response. Non-coding circular RNAs are ubiquitously expressed in mammalian tissue, highly conserved among species, and recently implicated in the regulation of macrophage activation. We sought to determine whether circRNAs modulate monocyte/macrophage biology and function.

We performed circRNA microarray analyses to assess transcriptome changes using RNA isolated from bone marrow derived macrophages polarized to a pro-inflammatory phenotype (INFγ + TNFα) or an anti-inflammatory phenotype (IL-10, IL-4, and TGF-β). Among differentially expressed circRNAs, circ-Cdr1as was chosen for further investigation. Additionally, we performed loss or gain of function studies to investigate if circ-Cdr1as is involved in phenotypic switching. For gain of function, we overexpressed circ-Cdr1as using pc3.1 plasmid with laccase2 flanking regions to promote circularization. For loss of function, we used a lentiviral short hairpin RNA targeting the circ-Cdr1as splicing junction.

Among circRNAs that are highly conserved and differentially expressed in pro- and anti-inflammatory lineages, circ-Cdr1as was one of the most downregulated in pro-inflammatory macrophages and significantly upregulated in anti-inflammatory macrophages in vitro. Overexpression of circ-Cdr1as increased transcription of anti-inflammatory markers and percentage of CD206+ cells in naïve and pro-inflammatory macrophages in vitro. Meanwhile, knockdown decreased transcription of anti-inflammatory markers and increased the percentage of CD86+ cells in naïve and anti-inflammatory macrophages in vitro.

This study suggests that circ-Cdr1as plays a key role in regulating anti-inflammatory phenotype of macrophages and may potentially be developed as an anti-inflammatory regulator in tissue inflammation.