Phenylacetylglutamine causes a pathologic inflammation state through the ß2-adrenergic receptor /cAMP/PKA/NF-κB pathway in diabetes

Abstract ID 54961 Poster Board 254 Background: Type 2 diabetes (T2DM) affects billions of patients and has a pathophysiologically important inflammatory component. Macrophage plasticity in diabetes is impaired, and remains in a persistent proinflammatory state; yet currently, the cause of the inflammatory state is unknown. Phenylacetylglutamine (PAGIn) is an amino acid metabolism end product that rises in T2DM patients. However, the role of PAGIn in the inflammatory component of T2DM remains unknown. Here, we aim to identify blood-based systemic factors for pathologic inflammation status and risk of chronic wounds as they are highly reproducible at low cost and search for effective and safe agents to attenuate diabetic inflammatory complications. Methods: In vivo, phenylacetylglutamine was dissolved in normal saline and injected in mice daily (intraperitoneal, 50mg/kg). In vitro, primary mice bone marrow-derived macrophages (BMDMs) were treated with phenylacetylglutamine (100 uM) for the indicated time. RNA-seq, Elisa, PCR, and western blot were used to characterize the impact of PAGln on BMDMs and mice wounds. Flow cytometry was taken to quantify and type immune cells in mice bone marrow, blood, spleens, and wounds. The activity of the NF-κB pathway was assessed by KEGG analysis, western blot, luciferase, and activity assay. Employing genetic and pharmacological tools, we detected the receptors through which PAGln mediates inflammatory events. Results: Here, increased PAGIn was identified in diabetes and in people suffering from chronic wounds in an independent cohort. Both conditions have been linked to a hyper-inflammatory state. In vivo, we found PAGln supplement resulted in worse wound healing and increased inflammatory gene expression in mice wounds. In vitro, PAGIn leads to macrophage M1 polarization. Mechanistically, we report that in wound healing, PAGIn increased in plasma upregulates cAMP levels in wound macrophages via ss2-adrenergic receptors and that this increase in cAMP induces NF-κB-mediated inflammatory gene transcription in wound macrophages via a PKA mechanism. Interestingly, we found that PAGln caused a significant increase in inflammatory macrophages in mice spleens, blood, bone marrow, as well as wounds and that this increase in inflammatory macrophages can be rescued by carvedilol (ß blocker). Conclusion: Our study unravels a novel mechanism by which PAGIn disturbs the transition of macrophage phenotype from inflammatory to reparative, facilitating pathologic inflammation, and impairing wound healing by activating the ss2-adrenergic receptor /cAMP/PKA/NF-κB pathway. The negative effect of PAGIn could be rescued by carvedilol (ss blocker). Thus, our recognition of chronic inflammation in diabetes needs to be revised, and understanding these mechanisms can inform alternative treatment strategies and refine chronic wound repair therapies for diabetic patients Funding: Shanghai Clinical Research Center of Plastic and Reconstructive Surgery supported by Science and Technology Commission of Shanghai Municipality (Grant No. 22MC1940300)