Monocytes Reprogrammed by 4-PBA Potently Contribute to the Resolution of Inflammation and Atherosclerosis

Background Chronic inflammation initiated by inflammatory monocytes underlies the pathogenesis of atherosclerosis. However, approaches that can effectively resolve chronic low-grade inflammation targeting monocytes are not readily available. The small chemical compound 4-phenylbutyric acid (4-PBA) exhibits broad anti-inflammatory effects in reducing atherosclerosis. Selective delivery of 4-PBA reprogrammed monocytes may hold novel potential in providing targeted and precision therapeutics for the treatment of atherosclerosis.Methods Systems analyses integrating single-cell RNA-sequencing and complementary immunological approaches characterized key resolving characteristics as well as defining markers of reprogrammed monocytes trained by 4-PBA. Molecular mechanisms responsible for monocyte reprogramming was assessed by integrated biochemical and genetic approaches. The inter-cellular propagation of homeostasis resolution was evaluated by co-culture assays with donor monocytes trained by 4-PBA and recipient naïve monocytes. The in vivo effects of monocyte resolution and atherosclerosis prevention by 4-PBA were assessed with the high fat diet-fed ApoE-/- mouse model with i.p. 4-PBA administration. Furthermore, the selective efficacy of 4-PBA trained monocytes were examined by i.v. transfusion of ex vivo trained monocytes by 4-PBA into recipient high fat diet-fed ApoE-/- mice.Results In this study, we found that monocytes can be potently reprogrammed by 4-PBA into an immune-resolving state characterized by reduced adhesion and enhanced expression of anti-inflammatory mediator CD24. Mechanistically, 4-PBA reduced the expression of ICAM-1 via reducing peroxisome stress and attenuating SYK-mTOR signaling. Concurrently, 4-PBA enhanced the expression of resolving mediator CD24 through promoting PPARγ neddylation mediated by TOLLIP. 4-PBA trained monocytes can effectively propagate anti-inflammation activity to neighboring monocytes through CD24. Our data further demonstrated that 4-PBA trained monocytes effectively reduce atherosclerosis pathogenesis when administered in vivo .Conclusion Our study describes a robust and effective approach to generate resolving monocytes, characterizes novel mechanisms for targeted monocyte reprogramming, and offers a precision-therapeutics for atherosclerosis based on delivering reprogrammed resolving monocytes.### Competing Interest StatementThe authors have declared no competing interest.* 4-PBA : 4-phenylbutyric acid BMM : bone marrow-derived monocyte CCL5 : Chemokine ligand 5 CFSE : Carboxyfluorescein succinimidyl ester H&E : Hematoxylin and Eosin HFD : high fat diet ICAM-1 : Intercellular adhesion molecule 1 M-CSF : Macrophage colony-stimulating factor m TOR : Mammalian target of rapamycin NEDD8 : Neural precursor cell expressed, developmentally down-regulated 8 OCT : optimal cutting temperature oxLDL : Oxidized low-density lipoprotein PPARγ : Peroxisome proliferator-activated receptor γ scRNA-seq : Single-cell RNA sequencing SYK : Spleen tyrosine kinase TOLLIP : Toll-interacting protein TRAM : Trif-related adapter molecule