Temporal mineralocorticoid receptor activation regulates the molecular clock and transcription of cardiovascular disease modulators in myeloid cells

Inappropriate mineralocorticoid receptor (MR) activation in monocytes/macrophages promotes cardiac inflammation and fibrosis. However, the specific pathways whereby the MR regulates macrophage phenotype are not fully defined. We recently identified bidirectional regulation of the MR and the molecular circadian clock in cardiac cells. Given that immune cells are important regulators of cardiac pathology, we investigated whether MR regulates the molecular circadian clock and time of day expression of inflammatory mediators in splenic monocytes/macrophages using myeloid MR null mice (MyMRKO). RNAseq and real-time quantitative PCR (RT-qPCR) analysis of whole spleen from floxed control (FC) or MyMRKO revealed differential expression of clock genes <i>Per2</i>, <i>Cry1</i>, <i>REV-ERBα</i>, and <i>DBP</i> at (Zeitgeber time) ZT0 versus ZT12. Time-of-day regulation of numerous gene targets was also disordered in MyMRKO spleen versus FC including <i>iNOS2</i>, <i>CXCR4</i>, <i>FABP3</i>, <i>S100A8</i> and <i>S100A9</i>, and <i>FGF1</i>. Aldosterone induction of <i>REV-ERBα</i>, <i>Cry1</i>, <i>iNOS</i>, <i>IL-1β</i>, <i>Arg-1</i>, <i>IL-10</i>, <i>CCL2</i>, and <i>Spp1</i> was greater when delivered at ZT0 versus ZT12, when corticosterone levels are low. Moreover, oscillating expressions of <i>Per2</i>, <i>REV-ERBα</i>, and other clock components were regulated by 10 nM aldosterone or corticosterone in immortalized bone marrow-derived cells, supporting a direct role for MR modulation of cellular clock time. Significant differences observed between male and female samples underscore the role of sex in the modulation of circadian signaling and MR-dependent pro-inflammatory phenotype in myeloid cells. Cardiac macrophage-specific bulk RNAseq and scRNAseq datasets verified MR-dependent regulation of many temporally induced genes in immune cell subsets, whereas FACS analysis showed that immune cell populations were mostly unchanged, and that <i>IL-1β</i> expression is highest in myeloid cells consistent with MyMRKO regulating <i>IL-1β</i> in this population. Our findings demonstrate the dynamic influence of MR transcriptional control of circadian clock and inflammatory pathways in myeloid cells, highlighting potential sex-based differences and offering insights into potential mechanisms underpinning MR modulation of myeloid cell phenotype.<b>NEW & NOTEWORTHY</b> Mineralocorticoid receptor (MR) signaling dynamically regulates the circadian clock and inflammatory gene expression in myeloid cells. Using myeloid-specific MR knockout mice, we identified disrupted time-of-day expression of core clock and inflammatory genes, with sex-based differences in response. These findings reveal novel MR-circadian clock interactions in immune cells and suggest a time- and sex-dependent mechanism by which MR shapes macrophage phenotype and potentially cardiac inflammation.