Macrophages signal cross-talks under hyperglycemic conditions and their link to cyclophilins

Abstract Background Hyperglycemia is associated with pro-inflammatory reprogramming of macrophages, increase of reactive oxygen species (ROS) levels and variations in ATP consumption. In addition, in these conditions there is a release of inflammatory proteins like cyclophilins (Cyps). This immunophilin family, which binds cyclosporine A (CsA) selectively, includes CypA, CypB and CypC, associated with inflammation, and CypD, linked to mitochondrial function. The aim of this work was to study the Cyps secretion profile by macrophages under high glucose (HG) conditions, the relationship with inflammatory pathways and the endoplasmic reticulum (ER). Methods The effects of HG were checked in RAW264.7 macrophages cell line. Western blot was used to quantify protein expression inside cells, in the extracellular medium and in extracellular vesicles (EVs). In addition, ROS, interleukin (IL-6) and ATP content were quantified by fluorescence, absorbance and luminescence dyes. Confocal microscopy was used to visualize calnexin. Results HG increased the intracellular expression of CypA and CypC while CypB was decreased. In these conditions, CypA, CypB and CypC were detected in the cell medium while CypD was absent. CypA and CypB were also identified within EVs after HG incubation, along with the Cyps-receptor CD147, upregulated under hyperglycemia. ERK1/2 and AMPK inflammation pathways, ROS, IL-6 and ATP levels were increased after HG treatment, effect attenuated by CsA-mediated Cyps inhibition. Since CypB and CypC are ER-resident proteins, ER modulation was analyzed by measuring pPERK, pEIF2⍺, pJNK, pNF-κβ p65 and GRP78 levels. The expression of these proteins was increased by hyperglycemia and this effect was reduced when Cyps were inhibited. Moreover, ER stimulation was confirmed by visualizing calnexin presence. Conclusions Our findings suggest that Cyps are involved in the pro-inflammatory response induced by HG in macrophages, potentially through AMPK activation and ER modulation. The presence of Cyps in EVs highlights a novel vesicle-mediated secretion mechanism under hyperglycemic conditions in these inflammatory cells. The current results strengthen the role of Cyps in inflammation and intercellular communication, suggesting a potential therapeutic target in inflammation-related disorders.