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. 2026 Jan 29;32:29. doi: 10.1186/s10020-026-01425-4

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

Noelia Castedo 1, Rebeca Alvariño 2, Amparo Alfonso 1,✉, Mercedes R Vieytes 2, Luis M Botana 1,✉
PMCID: PMC12924273  PMID: 41612185

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.

Supplementary Information

The online version contains supplementary material available at 10.1186/s10020-026-01425-4.

Keywords: Cyclophilins, Extracellular vesicles, Hyperglycemia, Inflammation, Macrophages

Background

Diet-related hyperglycemia represents a risk factor for developing many inflammatory-based diseases, such as cardiovascular diseases and diabetes, among others (Myette-Côté et al. 2018). The persistence of high glucose (HG) levels promotes a state of chronic inflammation with immune cells becoming activated (Edgar et al. 2021). In the vascular wall, monocytes recruitment is the first step on the chronic inflammatory processes, followed by monocyte transmigration, vascular permeability and monocyte differentiation into tissue macrophages. HG levels facilitate monocyte adhesion to endothelial cells and differentiation into macrophages (Ramachandran et al. 2016). Indeed, HG conditions induce pro-inflammatory reprogramming of macrophages, leading to the development of a persistent inflammatory state and the subsequent vascular complications (Rendra et al. 2019). Previous studies reported that HG promotes the secretion of several proteins from monocytes that aggravate the vascular lesion formation (Ramachandran et al. 2012). Among these proteins CypA is an immunophilin that increases lipid uptake in macrophages and promotes macrophage migration through endothelial cells (Ramachandran et al. 2018).

Cyclophilins (Cyps), also known as immunophilins, are peptidyl-prolyl cis/trans isomerase (PPIase) proteins which are involved in many inflammatory processes (Perrucci et al. 2015; Kumari et al. 2013). These proteins show affinity for the immunosuppressant drug cyclosporine A (CsA), a compound produced by the fungusTolyplocadium inflatum (Bukrinsky et al. 2015). Cyps are highly conserved molecular chaperones with 18 different isoforms located in many cellular compartments (Hoffmann and Schiene-Fischer 2014). This protein family includes CypA, CypB, CypC and CypD, among others. CypA is located in the cell cytosol and nucleus, and it is increased in microglia under inflammatory stimuli (Flora et al. 2019). CypA was also found elevated in serum from type 2 diabetes patients with coronary artery disease and has been associated to diabetic nephropathy progression (Gegunde et al. 2023; Chiu et al. 2018). CypB is an endoplasmic reticulum (ER)-resident protein that regulates the organelle homeostasis, playing a role in protein folding and protecting cells against ER activation (Perrucci et al. 2015). High CypB protein levels were found in serum from patients suffering from metabolic syndrome (Zhang et al. 2017). However, CypB’s role is controversial since it reduced cytokines secretion from lipopolysaccharide (LPS)-treated macrophages, elucidating a role in the inhibition of reactive oxygen species (ROS) production and inflammation (Marcant et al. 2012). Both CypA and CypB are released under inflammation, and they act as chemokines in the extracellular medium by binding to the membrane receptor CD147 (Perrucci et al. 2015; McClements et al. 2015). CD147 is a single-chain type I transmembrane protein from the immunoglobulin superfamily shown enhanced upon inflammatory conditions in neurons, glial and endothelium cells (Flora et al. 2019; Gegunde et al. 2021). CypC is also located in the ER and Golgi apparatus and modulates ER function (Bukrinsky 2015). This protein is less studied, but our group reported elevated CypC levels in the serum of coronary artery disease patients with increased blood glucose levels (Alfonso et al. 2019; Bayon et al. 2020). In addition, CypC was upregulated in rat brains in a model of brain ischemia, suggesting an important role of this protein in vascular and metabolic diseases (Shimizu et al. 2005). The interaction of CypC with CD147 remains undescribed but higher receptor levels were detected after CypC stimulation (Alvariño et al. 2022a). CypD is the only mitochondrial isoform and regulates the mitochondrial transition permeability pore opening (mPTP) (Amanakis and Murphy 2020). It was reported that deficiency of CypD decreases the inflammatory response in macrophages (Priber et al. 2015). Moreover, CypD modulation in microglia showed a reduction in cell activation (Alvariño et al. 2019).

Besides being found in the extracellular medium, Cyps were also detected in extracellular vesicles (EVs), with increased content of CypA, CypB and CypC inside EVs derived from BV2 microglia under HG (Castedo et al. 2025; Goetzl et al. 2021a; Wu et al. 2021). EVs, which contain DNA, RNA, proteins and other bioactive molecules, are membrane-bound structures of cellular topology with a diameter of 30–200 nm. The endosomal pathway is the main route of EVs generation, which are then released into the extracellular space where they play a crucial role in cell-to-cell communication (Pegtel and Gould 2019). EVs contain many transmembrane proteins used as surface signals, such as CD63, which mediates the inclusion of proteins that fluctuates with the inflammatory stimuli, since EVs serve as a mode of intercellular communication (Mathieu et al. 2021). In fact, macrophages release EVs during diabetes mellitus and obesity and the cargo of these vesicles is a leading factor to alter immune response but also a protective factor under these conditions (Kumar et al. 2022; Pardo et al. 2018).

Cyps appeared to play a crucial role in the inflammatory response of macrophages to different stimuli, a cell type that releases EVs in HG conditions. Indeed, previous results showed an increase of Cyps in microglia and in EVs derived from these cells under HG. Therefore, the objective of this work was to study the relationship of Cyps and their receptor with the inflammatory pathways activated by HG in macrophages. In this context, the expression of CypA, CypB, CypC, CypD and CD147 receptor and their content inside EVs under HG conditions in RAW264.7 murine macrophages was analyzed. In addition, the activation of inflammatory HG-related pathways and ER intracellular routes was also studied.

Materials and methods

Chemicals and solutions

D (+)-Glucose anhydrous (C6H12O6) was obtained from PanReac AppliChem (Barcelona, Spain) and CsA with a purity ≥ 98.5% from Abcam (Cambridge, UK). Polyvinylidene difluoride (PVDF) membrane, bovine serum albumin, LPS from Escherichia coli O111:B4 and the 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyl tetrazolium bromide (MTT) dye were purchased from Sigma-Aldrich (Madrid, Spain). Total Exosome Isolation Reagent, Exosome-Depleted Fetal Bovine Serum and 5-(and 6-)-carboxy-2’,7’-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA) were purchased from Thermo Fisher Scientific (Madrid, Spain). Polyacrylamide gels and molecular weight marker Precision Plus Protein Standards Kaleidoscope were obtained from Bio-Rad (Barcelona, Spain). Protease Inhibitor Complete Tablets and Phosphatase Inhibitor Cocktail Tablets were from Roche (Madrid, Spain). The composition of the Locke’s Buffer used in the viability assays was (in mM) 154 NaCl, 5.6 KCl, 1.3 CaCl2, 1 MgCl2, 3.6 NaHCO3, 5.6 Glucose and 10 HEPES. The RIPA buffer used on the EVs lysis was composed by 150 mM sodium chloride, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate (SDS), 50 mM Tris pH 8.0. The composition of the lysis buffer used to obtain the cytosolic and membrane fractions was (in mM) 50 Tris–HCl (pH 7.4), 150 NaCl, 1 EDTA and 1% Triton X-100, containing phosphatase/protease inhibitors cocktails. The composition of the standard reaction solution used in the ATP assays was 4.45 mL H2O, 250 μL 20X reaction buffer, 50 μL 0.1 M DTT, 250 μL 10 mM luciferin and 1.25 μL luciferase. The phosphate buffered saline (PBS) solution was composed by: 137 mM NaCl, 8.2 mM Na2HPO4, 1.5 mM KH2PO4 and 3.2 mM KCl. ELISA Kit and ATP Determination Kit were purchased from Thermo Fisher Scientific (Madrid, Spain).

Cell culture

Murine macrophage RAW264.7 cell line was purchased from American Type Culture Collection, number TIB-71. Cells were maintained in DMEM low glucose (1 g/L) medium with L-Glutamine, phenol red and sodium pyruvate (110 mg/L) at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. It was supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 μg/mL). Cells were mechanically dissociated twice a week using a scrapper. All the reagents were provided by Thermo Fisher Scientific.

Cell viability assay

Cells were seeded in 96-well plates at a density of 5 × 104 cells per well. After 24 h, cells were treated with different HG concentrations (10–50 mM) and pretreated 1 h with CsA (1 μM), if applicable. The MTT dye was used to determine the effect of HG on cell viability. After 24 h of treatment, cells were washed three times with Locke’s/saline buffer, and 200 µL MTT (500 µg/mL) dissolved in the saline buffer were added to each well. After 1 h of incubation at 37 °C and 300 rpm, cells were disaggregated with 5% SDS. A spectrophotometer plate reader was used to measure formazan crystal formation at 595 nm. Saponin at 1 mg/mL was used as death control. Experiments were performed in triplicate at least on three independent times.

Protein extraction

A total of 2 × 106 cells per well were seeded in 6-well plates and treated with HG for 24 h. Cells were washed twice with ice-cold PBS and 100 μL lysis buffer were added to each well to obtain the cytosolic protein fraction. Cells were centrifuged 15 min at 4 °C and 3000 rpm. Supernatant was collected as the cytosolic fraction and the pellet was resuspended on 30 μL lysis buffer and incubated on ice for 30 min with intervals of sonication. Subsequently, the pellet resuspended in lysis buffer was centrifuged 30 min at 4 °C and 12,000 rpm, and the membrane fraction was collected. Protein quantification was performed using the Direct Detect system (Merck Millipore).

Extracellular vesicles isolation and purification

RAW264.7 cells were seeded at a density of 2 × 106 cells per well in 6-well plates. After 24 h of treatment with HG, the supernatant was centrifuged for 30 min at 4 °C and 3100 rpm and then filtered and concentrated through 0.22 μM filters first and, then past ultracentrifuge tubes (Amicon Ultra Centrifugal Filter, 30 kDa MWCO from Sigma-Aldrich). The supernatant obtained was incubated overnight with the Total Exosome Isolation Reagent (500 µL of reagent per milliliter of supernatant) and then centrifuged for 1 h at 4 °C and 13,000 rpm. Supernatant was discarded and the EVs pellet was resuspended in 60 μL PBS and stored at—20 °C. For protein analysis, 60 μL of RIPA buffer containing phosphatase/protease inhibitors were added to isolated EVs. Protein concentration was determined by Bradford method after centrifugation at 4 °C and 6000 rpm for 10 min. Experiments were performed three independent times.

To collect the protein contained in the cell medium, the filtrate obtained from Amicon 30 kDa filtration was transferred to Amicon 10 kDa ultracentrifuge filters and centrifuged 10 min at 4 °C and 4000 rpm. The concentrate was stored at—20 °C.

TEM imaging and NTA characterization of extracellular vesicles

For visualization by transmission electron microscopy (TEM), purified EVs were fixed with 2% paraformaldehyde. Briefly, 1 mL of 2% paraformaldehyde was added to the pellet of EVs and left for 5 min. Samples were then centrifuged at 4 °C and 13,000 rpm for 1 h. The supernatant was discarded and 100 μL of PBS were added before preparation for TEM.

Nanoparticle Tracking Analysis (NTA) was used to characterize EVs size and concentration from RAW264.7 cells by measuring the movement of EVs through image analysis. Briefly, 1 ml of the EVs pellet-PBS resuspended in RNase-free water (1:1000 dilution factor) was injected into the NanoSight NS300 instrument (Malvern, UK) equipped with a 488 nm blue laser and a sCMOS camera. NanoSight software (NTA 3.4 Build 3.4.003) was used for sample analysis and recording of five 60-s videos with the following settings: syringe pump speed 40, camera level 14, temperature 25 °C and viscosity 0.9 cP (water). The analysis settings were: detection threshold 16 and the focus was manually adjusted.

Western blot analysis

Western blotting of EVs, cell supernatant and cytosolic or membrane protein content was performed after protein extraction. In brief, 15 μg of EVs content, 10 μg of cytosolic and membrane protein or 15 μL of filtered culture supernatant were resolved on a 4–20% polyacrylamide gel. The proteins were transferred to PVDF membranes and subjected to electrophoresis using the Trans-Blot semi-dry transfer system. The molecular weight marker Precision Plus Protein Standard Kaleidoscope was used for protein weight determination. Briefly, membranes were blocked with 0.5% BSA and incubated with antibodies using the SNAP i.d. system (Merck). CypA was detected with anti-PPIA primary antibody (1:1000, Elabscience), CypB was quantified with anti-PPIB (1:1000, Elabscience), CypC was identified with anti-PPIC (1:1000, Elabscience) and CypD was distinguished with anti-cyclophilin F primary antibody (1:1000, Abcam). CD147 receptor was detected with anti-CD147 (1:10,000, Abcam), pPERK with anti-pPERK (1:500, Santa Cruz Biotechnology) and pEIF2⍺ with anti-pEIF2⍺ (1:200, Santa Cruz Biotechnology). ERK and pERK were identified with anti-ERK (1:1000, Thermo Fisher) and anti-pERK (Thr202/Tyr204, Thr185/Tyr187) (1:1000, Sigma-Aldrich), respectively. GSK3β and pGSK3β were observed with anti-GSK3β (1:1000, Sigma-Aldrich) and anti-pGSK3β (Ser9) (1:1000, Sigma-Aldrich), AMPK and pAMPK with anti-AMPK (1:500, Sigma-Aldrich) and anti-pAMPK (Thr172) (1:500, Sigma-Aldrich). JNK and pJNK were identified with anti-JNK (1:1000, BD Biosciences) and anti-pJNK (Thr183, Tyr185) (1:1000, BD Biosciences), respectively. NF-κβ p65 and pNF-κβ p65 were detected with anti- NF-κβ p65 (1:1000, Abcam) and anti-pNF-κβ p65 (Ser536) (1:1000, Abcam). Anti-GRP78 (1:10,000, Sigma-Aldrich) was used for detecting GRP78. The specificity of Cyps antibodies was previously tested (Alvariño et al. 2022b). The detection of specific protein bands was performed using Supersignal West Pico or Supersignal West Femto. The intensity of the protein bands was normalized using anti-GAPDH (1:1000, Sigma-Aldrich) in the cytosolic lysates, anti-CD63 (1:1000, Abcam) in the EVs lysates and anti-Sodium/Potassium ATPase (1:10,000, Abcam) in the membrane fraction. Protein bands were densitometrically analyzed using Diversity GeneSnap (Syngene). All measurements were performed in duplicate on three independent experiments. The original western blots images are in the Additional File 1.

ATP assays

ATP content inside cells was determined through the use of an ATP Determination Kit. Briefly, 5 × 104 cells per well were seeded in 96-well plates. After 24 h of treatment with HG, cells were washed once with PBS and 200 μL of lysis buffer (PBS—1% Triton X100) were added to each well. In a white well plate, 100 μL of the standard reaction solution and 10 μL of each sample were added to each well. Then, after 15 min of incubation at 150 rpm and room temperature in the dark, luminescence was measured. A standard curve was used to quantify ATP levels inside macrophages. All measurements were performed in triplicate at least three independent times.

ROS assays

Intracellular ROS levels were determined with carboxy-H2DCFDA dye, as previously described (Alvariño et al. 2019). A total of 5 × 104 cells per well were seeded in 96-well plates for 24 h at 37 °C. After 24 h of treatment with HG, cells were washed twice with DMEM low glucose medium without FBS and 200 μL of 20 μM carboxy-H2DCFDA were added to each well. After 1 h of incubation at 37 °C, the dye was removed and 200 μL of PBS were added. Cells were incubated for 30 min at 37 °C, and ROS levels were measured at 495 nm excitation and 527 nm emission with a spectrophotometer plate reader. All measurements were performed in triplicate at least three independent times.

IL-6 secretion

The release of interleukin-6 (IL-6) by macrophages was measured in the cell culture supernatant with an ELISA kit following manufacturer’s instructions. RAW264.7 macrophages were seeded in 96-well plates at a density of 5 × 104 cells per well for 24 h. Cells were incubated 24 h under HG conditions. All samples were run in duplicate and cytokine concentration was calculated with a standard curve. The optical density of each well was obtained with a spectrophotometer microplate reader at a wavelength of 450 nm.

Calnexin staining

ER stress was assessed as calnexin detection by confocal microscopy. Briefly, after 24 h of treatment under HG, cells were washed with PBS and fixed with 4% paraformaldehyde at room temperature for 10 min. Then, cells were incubated 2 h with PBS-5% BSA- 0.1% Triton X-100 followed by an overnight incubation with anti-calnexin (1:1000, Abcam) primary antibody at 4 °C. Afterwards, cells were incubated with anti-Cy3 goat anti-rabbit IgG (1:1000, Invitrogen) secondary antibody for 2 h at room temperature. Finally, Oregon Green 488 (0.165 μM) was added for 20 min at room temperature to stain the F-actin skeleton. Then, coverslips were mounted in glycerol-PBS (1:1).

Images were acquired with a 40X oil immersion objective in a Nikon Eclipse TE2000-E inverted microscope attached to the C1 laser confocal system (EZC1 V.2:20 software; Nikon Instruments Europe B.V., Netherlands). To excite the Cy3 antibody, a 561 nm helium–neon laser was used and Oregon Green dye was excited by a 488 nm laser argon. Fluorescent images were acquired at a resolution of 512 × 512 pixels separately for each fluorophore and then mixed to avoid interferences. No interferences of Cy3 in green channel were observed, as well as no interferences of Oregon Green in red channel were detected.

Statistical analyses

Data are presented as mean ± SEM. Statistical differences were evaluated by one way ANOVA with Dunnett’s or Tuckey’s post hoc test using the GraphPad Prism v.10 software. Statistical significance was considered at p < 0.05.

Results

High Cyps levels were present in the serum from patients suffering from cardiovascular and metabolic diseases associated with diabetes (Gegunde et al. 2023; Zhang et al. 2017; Alfonso et al. 2019). In these patients, lymphocytes contained an increased Cyps expression. In addition, high Cyps content was detected in microglia cells and EVs derived from microglia under HG conditions (Castedo et al. 2025). Nevertheless, there is no data available regarding CypA, CypB, CypC and CypD levels and their content inside EVs in peripheric cells under HG. In this context, the aim of this work was to determine the profile of Cyps and the activation of inflammatory pathways in the context of Cyps under HG in macrophages.

First, the effect of HG treatment on macrophage viability was analyzed through MTT assay. RAW264.7 cells were seeded on DMEM low glucose medium with 5.5 mM glucose. In those conditions, cells were treated with higher glucose concentrations (10–50 mM) for 24 h to observe the effect of HG on the viability of the macrophages. LPS (1 μg/mL) was used as positive control of inflammation. None of HG treatments reduced cell viability of macrophages compared to the control cells with 5.5 mM glucose (Supplementary Fig. 1 in Additional File 2).

The next step was to determine the role of Cyps in the inflammatory response underlying HG, so the intracellular expression of CypA, B, C and D was analyzed by western blot. In that way, 25 mM glucose (450 mg/dL) and 50 mM glucose (900 mg/dL) were the concentrations chosen to perform the next set of experiments. In microglia cells, 25 mM of glucose induces a severe inflammatory response associated to Cyps levels (Castedo et al. 2025). Therefore, this concentration was initially selected to perform experiments in addition to 50 mM of glucose that is considered a severely uncontrolled hyperglycemic state in macrophages (Ghandour et al. 2024). These glucose concentrations did not induce a toxic effect in macrophages and cover a wide range of hyperglycemia conditions. Thus, from now, HG will include 25 mM and 50 mM glucose. When RAW264.7 cells were incubated with HG, the intracellular expression of CypA was significantly increased by both 25 mM (116.97 ± 0.7%) and 50 mM glucose (120.28 ± 6.12%) compared to control cells (Figs. 1a and 1b). In the same conditions, inhibition by CsA (1 μM) reduced CypA macrophage expression under 25 mM (106.64 ± 1.66%) and 50 mM (111.47 ± 8.78%). When intracellular CypB expression was analyzed, a significant decrease was detected by 25 mM and 50 mM glucose treatment compared to control cells (41.89 ± 3.14% and 59.13 ± 9.61%, respectively) (Figs. 1c and 1 d). Under 50 mM glucose, CsA pretreatment significantly reduced CypB expression (26.38 ± 5.81%) (Fig. 1d). Moreover, CypB intracellular levels were decreased to 30% by CsA (Figs. 1c and 1 d). If compared to CypA, the increased expression of CypB elicited by LPS, to mimic an acute inflammatory stimulus, is remarkable for both glucose levels (124.82 ± 1.63% and 178.59 ± 33.15%, respectively) (Fig. 1). Inhibition of Cyps by CsA under LPS treatment, diminished CypA expression by 10% and CypB expression to 70% (Fig. 1). Likewise, the inhibitory effect of CypB expression caused by CsA is very high and opposed to the effect of LPS (32.31 ± 6.14% CsA vs 174.29 ± 11.07% LPS) (Figs. 1c and 1 d). CypC intracellular expression was significantly increased in 25 mM glucose (165.29 ± 6.80%) and CsA pretreatment reduced CypC almost at control levels (Fig. 2a). In contrast, 50 mM glucose reduced CypC expression (66.33 ± 12.54%) compared to control cells (Fig. 2b). In these conditions, CsA pretreatment did not affect CypC levels. CypD intracellular expression was unaffected by HG treatment compared to control cells (Figs. 2c and d).

Fig. 1.

Fig. 1

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Intracellular expression of CypA and CypB under HG conditions in RAW264.7 cells. CypA expression under 25 mM (a) and 50 mM (b) glucose. CypB expression under 25 mM (c) and 50 mM (d) glucose. Cells were stimulated with 25 mM and 50 mM glucose for 24 h and pretreated with 1 μM CsA for 1 h, if applicable. LPS at 1 μg/mL was used as positive control of inflammation. Band intensity was normalized by GAPDH. Data are mean ± SEM of three independent replicates performed by duplicate. Data are expressed as percentage of control cells (5.5 mM glucose). Statistical differences determined by One way ANOVA test and Tuckey’s post hoc test. *p < 0.05, **p < 0.01 and ***p < 0.001 compared to control cells. # p < 0.05 between groups

Fig. 2.

Fig. 2

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Intracellular expression of CypC and CypD after HG incubation in RAW264.7 macrophages. CypC expression under 25 mM (a) and 50 mM (b) glucose. CypD expression under 25 mM (c) and 50 mM (d) glucose. Cells were stimulated with 25 mM and 50 mM glucose for 24 h and pretreated with 1 μM CsA for 1 h, if applicable. LPS at 1 μg/mL was used as positive control of inflammation. Band intensity was normalized by GAPDH. Data are mean ± SEM of three independent replicates performed by duplicate. Data are expressed as percentage of control cells (5.5 mM glucose). Statistical differences determined by One way ANOVA test and Tuckey’s post hoc test. *p < 0.05 and **p < 0.01 compared to control cells. # p < 0.05 between groups

Since some Cyps intracellular expression was reduced, and these proteins were found increased in serum from patients, the following step was to analyze Cyps release to the extracellular medium by HG treatment (Gegunde et al. 2023). Cyps secretion was evaluated by western blot and both CypA and CypB were present in the medium of RAW264.7 cells under HG and LPS treatment (Supplementary Figs. 2a and 2b in Additional File 2). Moreover, under 50 mM glucose incubation, CypC was also released to the extracellular medium (Supplementary Fig. 2b in Additional File 2). CypD was not detected in the medium under any treatment condition.

As CD147 is the primary receptor for both extracellular CypA and CypB and it is externalized to the cell surface upon detection of elevated Cyp levels, the expression of glycosylated (active form) and non-glycosylated (inactive form) CD147 inside macrophages and on cell surface was analyzed. In the presence of 25 mM glucose, active CD147 receptor was translocated to the membrane (138.23 ± 11.85%), an effect reduced when Cyps were inhibited (100.63 ± 0.90%) (Fig. 3a). In contrast, in the cytosol fraction, glycosylated CD147 was significantly decreased by 25 mM glucose (80.48 ± 7.06%), as for the other treatments (Fig. 3b). The inactive form of CD147 was undetected on the membrane under 25 mM glucose while it was significantly enhanced by an inflammatory stimulus (LPS) (316.84 ± 8.66%) (Fig. 3c). When combined with LPS, CsA pretreatment significantly reduced the non-glycosylated CD147 expression on the membrane of macrophages (107.76 ± 21.10%) (Fig. 3c). In the cytosol, inactive CD147 was significantly decreased under 25 mM glucose (44.86 ± 16.19%) while LPS enhanced the expression of the receptor (179.80 ± 5.37%) (Fig. 3d). Interestingly, when Cyps were inhibited in macrophages treated with LPS, non-glycosylated CD147 levels were significantly reduced (105.44 ± 6.14%) (Fig. 3d). Treatment with 50 mM glucose also enhanced the expression of the glycosylated receptor in the membrane (160.32 ± 15.31%) (Fig. 3e), which was reduced by CsA (81.69 ± 0.15%) (Fig. 3e). As mentioned before, LPS reduced glycosylated CD147 expression in the cell cytosol (72.46 ± 6.46%) (Fig. 3f) compared to control cells. When analyzing the inactive form of CD147 on the cell membrane, no receptor presence was detected under 50 mM glucose (Fig. 3g). In contrast, LPS incubation increased the expression of the inactive receptor, which was reduced in combination with CsA (Fig. 3g), as previously reported. In the cell cytosol, the non-glycosylated form of CD147 was significantly reduced under 50 mM glucose (58.29 ± 9.07%) (Fig. 3h) and when combined with CsA no effect was observed. However, LPS stimulus enhanced inactive CD147 levels, reduced by CsA pretreatment (Fig. 3h), as aforementioned. Taking all together, HG treatment induces CypA and CypC expression and activates CD147, while LPS stimulation promotes the expression of CypA and CypB but inactivates CD147. In that way, it seems that the inflammatory response induced by HG is different from the response observed under LPS at 24 h.

Fig. 3.

Fig. 3

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Effect of HG on CD147 intracellular and surface expression in RAW264.7 macrophages. Glycosylated CD147 expression under 25 mM in the membrane (a) and in the cytosol (b). Non-glycosylated CD147 expression under 25 mM in the membrane (c) and in the cytosol (d). Glycosylated CD147 expression under 50 mM in the membrane (e) and in the cytosol (f). Non-glycosylated CD147 expression under 50 mM in the membrane (g) and in the cytosol (h). Cells were stimulated with 25 mM and 50 mM glucose for 24 h and pretreated with 1 μM CsA for 1 h, if applicable. LPS at 1 μg/mL was used as positive control of inflammation. Band intensity was normalized by GAPDH in the cytosol samples and Na/K-ATPase in the membrane fraction. Data are mean ± SEM of three independent replicates performed by duplicate. Data are expressed as percentage of control cells (5.5 mM glucose). Statistical differences determined by One way ANOVA test and Tuckey’s post hoc test. *p < 0.05, **p < 0.01 and ***p < 0.001 compared to control cells. # p < 0.05 and ### p < 0.001 between groups

Since macrophages release EVs under HG and Cyps were detected inside microglia-derived EVs in the context of elevated glucose, the levels of Cyps inside EVs from macrophages were analyzed (Castedo et al. 2025; Zhu et al. 2020). First, EVs release from RAW264.7 cells was characterized by TEM imaging and laser NTA (Supplementary Figs. 3a and 3b in Additional File 2), and it was observed a homogenous population of EVs with a similar concentration of particles and size to glial inflammatory cells (Castedo et al. 2025). The average vesicles concentration was 8e + 11 particles/mL and the mean particle size was around 165 nm. Therefore, Cyps content inside these vesicles was analyzed (Fig. 4). CypA content inside EVs derived from RAW264.7 cells was significantly reduced under 25 mM glucose (47.89 ± 15.78%) (Fig. 4a). This decrease was higher when cells were pretreated with CsA. In contrast, LPS enhanced CypA content (133.41 ± 9.49%), which was reduced by CsA (76.71 ± 17.46%) (Fig. 4a). When macrophages were incubated with 25 mM, CypB EVs levels were unaffected compared to control cells, an effect observed under the acute inflammatory stimulus (Fig. 4b). Surprisingly, when macrophages were treated with CsA alone and combined with glucose and LPS, CypB content inside EVs was significantly increased (482.06 ± 129.25% 25 mM and 286.51 ± 79.63% LPS, respectively). When analyzing CD147 content, 25 mM glucose significantly enhanced CD147 secretion through EVs (127.12 ± 10.61%) (Fig. 4c). In the same conditions, Cyps inhibition significantly reduced CD147 content inside EVs (56.33 ± 6.63%) (Fig. 4c). CD147 levels in vesicles were also enhanced by 24 h of LPS treatment (130.60 ± 16.59%), an effect mitigated by CsA (60.42 ± 7.42%) (Fig. 4c). The same results were obtained under 50 mM glucose incubation (Supplementary Fig. 4 in Additional File 2). CypC and CypD were not detected inside EVs from macrophages under any of the treatment conditions. In summary, the inflammatory response elicited by HG causes EVs to increase the levels of CypB and CD147, while LPS-mediated inflammation increases CypA and CD147 in EVs. Oddly, the response to CsA is opposite for CypA (decrease) and CypB (increase).

Fig. 4.

Fig. 4

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Cyps content inside EVs derived from macrophages under HG conditions. a CypA, b CypB and c CD147 levels in EVs. Cells were stimulated with 25 mM for 24 h and pretreated with 1 μM CsA for 1 h, if applicable. LPS (1 μg/mL) was used as positive control of EVs release and CsA as internal control of Cyps route. Band intensity was normalized by CD63, a surface EVs protein. Data are mean ± SEM of three independent replicates. Data are expressed as percentage of control cells. Statistical differences determined by One way ANOVA test and Tuckey’s post hoc test. *p < 0.05, **p < 0.01 and ***p < 0.001 compared to control cells. # p < 0.05 and ## p < 0.01 between groups

As previously shown, HG treatment activates CD147 receptor and when activated, extracellular Cyps stimulate several transduction pathways, such as extracellular signal-regulated kinase 1/2 (ERK1/2) and glycogen synthase kinase-3β (GSK3β) (Marcant et al. 2012; Kim et al. 2012; Boulos et al. 2007). In addition, ERK1/2 activation has been related to increased CypA and CypB levels and to the blockage of CypD-mediated mPTP opening (Marcant et al. 2012; Boulos et al. 2007; Porter and Beutner 2018; Pakula et al. 2007). Moreover, when CypA or CypB are inhibited, the phosphorylation of residues implicated in ERK1/2 activation is reduced (Kim et al. 2012; Li et al. 2020). Thus, the phosphorylation state of the residues involved in ERK1/2 activation and in GSK3β inhibition together with their total amounts were determined. Under HG treatment, phosphorylated ERK1/2 was decreased (25 mM: 62.86 ± 6.02%; 50 mM: 46.42 ± 13.62%), which means that ERK1/2 route is inactivated by HG (Figs. 5a and 5b). In contrast, this pathway was significantly activated by the inflammatory stimulus (LPS) (447.86 ± 66.96%). In the same conditions, supression of Cyps had no effect in the activation of this route, but CsA alone significantly reduced pERK/ERK ratio compared to control cells (70.90 ± 5.89%). Finally, the inactivation of GSK3β route was analyzed and it resulted unaffected by any of the treatments assayed (Figs. 5c and 5 d).

Fig. 5.

Fig. 5

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Effect of HG stimulation on the intracellular levels of pERK/ERK and pGSK3β/GSK3β. Intracellular expression of pERK/ERK under 25 mM (a) and 50 mM (b) glucose. Intracellular expression of pGSK3β/GSK3β under 25 mM (c) and 50 mM (d) glucose. Cells were stimulated with 25 mM and 50 mM glucose for 24 h and pretreated with 1 μM CsA for 1 h, if applicable. LPS at 1 μg/mL was used as positive control of inflammation. Band intensity was normalized by GAPDH. Data are mean ± SEM of three independent replicates performed by duplicate. Data are expressed as percentage of control cells (5.5 mM glucose). Statistical differences determined by One way ANOVA test and Tuckey’s post hoc test. *p < 0.05, **p < 0.01 and ***p < 0.001 compared to control cells

Considering the results obtained with ERK1/2 route, the activation of AMP-activated protein kinase (AMPK) was analyzed, since there is an inhibitory crosstalk between these two pathways. AMPK is an energy sensor targeted in the treatment of metabolic syndrome that monitors intracellular energy balance and regulates whole-body energy metabolism (Carling 2017). Under 25 mM glucose treatment, pAMPK/AMPK ratio was significantly increased compared to control cells (120.94 ± 4.13%) (Fig. 6a). Surprisingly, when Cyps were inhibited by CsA, AMPK phosphorylation was significantly reduced compared to 25 mM glucose treatment (101.61 ± 0.24%) (Fig. 6a). In addition, incubation with 50 mM glucose induced a huge activation of AMPK (195.63 ± 18.07%) and inhibition of Cyps reduced it significantly (86.28 ± 23.73%) (Fig. 6b). In contrast, the inflammatory stimulus (LPS) reduced the activation of AMPK pathway compared to control cells (77.09 ± 6.98%) and CsA pretreatment had no effect (Figs. 6a and 6b). Activation of AMPK increases the rate of catabolic pathways (ATP-generating) and decreases the rate of anabolic pathways (ATP-consuming) (Carling 2017; Vahidi Ferdowsi et al. 2021; Schultze et al. 2012). Therefore, ATP content inside RAW264.7 cells was measured. Under both 25 mM and 50 mM glucose incubation, ATP levels were significantly increased (6.86 ± 1.30 μM and 6.68 ± 0.36 μM, respectively) compared to control cells (2.08 ± 0.14 μM). In these treatment conditions, Cyps inhibition by CsA decreased ATP levels under both 25 mM (4.78 ± 0.48 μM) and 50 mM glucose (3.92 ± 0.09 μM). In contrast, LPS significantly decreased ATP content inside macrophages compared to control cells (1.11 ± 0.07 μM).

Fig. 6.

Fig. 6

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Effect of HG stimulation on pAMPK/AMPK intracellular expression and ATP content in RAW264.7 cells. Intracellular expression of pAMPK/AMPK under 25 mM (a) and 50 mM (b) glucose and ATP content inside macrophages after HG treatment (c). Cells were stimulated with 25 mM and 50 mM glucose for 24 h and pretreated with 1 μM CsA for 1 h, if applicable. LPS at 1 μg/mL was used as positive control of inflammation. Band intensity was normalized by GAPDH. Data are mean ± SEM of three independent replicates performed by duplicate. Data are expressed as percentage of control cells (5.5 mM glucose). Statistical differences determined by One way ANOVA test and Tuckey’s post hoc test. *p < 0.05, **p < 0.01 and ***p < 0.001 compared to control cells. # p < 0.05 and ## p < 0.01 between groups

Since AMPK is related to the modulation of ER, which is the main reservoir of CypB and CypC, the relationship between Cyps and the ER under HG was studied (Grenier et al. 2022; Stocki et al. 2014). Therefore, the expression of ER activation markers, namely phosphorylated protein kinase RNA-like endoplasmic reticulum kinase (pPERK) and phosphorylated eukaryotic translation initiation factor 2⍺ (pEIF2⍺) was determined. Thapsigargin (TG) (1 μM), a non-competitive inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), was used as a positive control. The expression of pPERK was significantly increased under 25 mM glucose (232.73 ± 22.91%), while under 50 mM glucose PERK was not activated compared to control cells (94.89 ± 5.70%) (Figs. 7a and 7b). Nevertheless, pEIF2⍺ intracellular expression was unaffected by 25 mM glucose (98.94 ± 12.36%), while it was significantly increased by 50 mM glucose (213.88 ± 45.51%) (Figs. 7c and 7 d). Cyps supression by CsA significantly decreased the expression of both pPERK (110.16 ± 26.62%) and pEIF2⍺ (85.27 ± 23.36%) under 25 mM and 50 mM glucose, respectively. LPS stimulus also enhanced pPERK (170.74 ± 24.72%) and pEIF2⍺ intracellular expression (249.72 ± 4.86%). In these conditions, inhibition of Cyps reduced the ER markers levels to 70% and 110%, respectively. TG positive control significantly increased both proteins expression (pPERK: 137.29 ± 4.65%; pEIF2⍺: 191.93 ± 52.74%). In summary, while the response to LPS is identified as inflammatory in all conditions, there seems to be a triggering signal that is conditioned by the level of glucose and functions in opposite directions for pPERK or pEIF2⍺.

Fig. 7.

Fig. 7

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Effect of HG treatment on the expression of the ER sensors pPERK and pEIF2⍺. Intracellular expression of pPERK under 25 mM (a) and 50 mM (b) glucose and intracellular levels of pEIF2⍺ under 25 mM (c) and 50 mM (d) glucose. Cells were stimulated with 25 mM and 50 mM glucose for 24 h and pretreated with 1 μM CsA for 1 h, if applicable. LPS at 1 μg/mL was used as positive control of inflammation and TG 1 μM as positive control of ER modulation. Band intensity was normalized by GAPDH. Data are mean ± SEM of three independent replicates performed by duplicate. Data are expressed as percentage of control cells (5.5 mM glucose). Statistical differences determined by One way ANOVA test and Tuckey’s post hoc test. *p < 0.05, **p < 0.01 and ***p < 0.001 compared to control cells. # p < 0.05 between groups

In view that HG induces the activation of the ER branch pPERK- pEIF2⍺, the effect of glucose over other ER stress routes was analyzed. First, the activation of jun N-terminal kinase (JNK) was determined, since it is a downstream target of endoribonuclease inositol-requiring enzyme 1α (IRE1α) (Li et al. 2024). Thus, the phosphorylation state of the residues involved in JNK activation, together with their total amounts were determined. Under HG treatment, JNK was activated since the ratio of the active form was increased (25 mM: 125.40 ± 2.42%; 50 mM: 195.13 ± 33.56%) (Figs. 8a and 8b). In addition, LPS stimulus activated this pathway (331.57 ± 28.66%), as well as the TG positive control (237.87 ± 28.80%) (Fig. 8). In the same conditions, inhibition of Cyps by CsA reduced the stimulation of this route, being significant only under 25 mM glucose (98.20 ± 8.38%). The activation of the JNK pathway promotes nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κβ) signaling (Abekura et al. 2019). This pathway is also triggered when the activating transcription factor 6 (ATF6) axis of the ER prompting response is stimulated (Stengel et al. 2020). Therefore, to determine whether HG also engages both axes, we examined the levels of the NF-κβ p65 subunit. Under HG conditions, both the phosphorylated (25 mM: 125.40 ± 2.42%; 50 mM: 450.82 ± 48.31%) (Figs. 9a and 9b) and the total forms of NF-κβ p65 (25 mM: 686.54 ± 91.88%; 50 mM: 452.93 ± 98.22%) were increased (Figs. 9c and 9 d). A similar effect was observed following LPS stimulation, while treatment with the TG control only enhanced the total amount of the transcription factor without promoting its phosphorylation (Fig. 9). Notably, in these conditions, Cyps suppression reduced both phosphorylated and total p65 levels, indicating again a role of Cyps in ER stress.

Fig. 8.

Fig. 8

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Effect of HG treatment on the expression of JNK. Intracellular expression of pJNK and JNK under 25 mM (a) and 50 mM (b) glucose. Cells were stimulated with 25 mM and 50 mM glucose for 24 h and pretreated with 1 μM CsA for 1 h, if applicable. LPS at 1 μg/mL was used as positive control of inflammation and TG 1 μM as positive control of ER modulation. Band intensity was normalized by GAPDH. Data are mean ± SEM of three independent replicates performed by duplicate. Data are expressed as percentage of control cells (5.5 mM glucose). Statistical differences determined by One way ANOVA test and Tuckey’s post hoc test. *p < 0.05 and **p < 0.01 compared to control cells. # p < 0.05 between groups

Fig. 9.

Fig. 9

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Intracellular expression of pNF-κβ and NF-κβ after HG stimulation. pNF-κβ levels under 25 mM (a) and 50 mM (b) glucose and intracellular levels of NF-κβ under 25 mM (c) and 50 mM (d) glucose. Cells were stimulated with 25 mM and 50 mM glucose for 24 h and pretreated with 1 μM CsA for 1 h, if applicable. LPS at 1 μg/mL was used as positive control of inflammation and TG 1 μM as positive control of ER modulation. Band intensity was normalized by GAPDH. Data are mean ± SEM of three independent replicates performed by duplicate. Data are expressed as percentage of control cells (5.5 mM glucose). Statistical differences determined by One way ANOVA test and Tuckey’s post hoc test. *p < 0.05 and **p < 0.01 compared to control cells. # p < 0.05 between groups

The ER prompting responses showed above induce the transcription of ER folding proteins, such as glucose-regulated proteins (GRPs), as an adaptive action (Ibrahim et al. 2019). Under ER stimulation, glucose-regulated protein 78 (GRP78) is removed from PERK, IRE1α and ATF6, and the three axes are triggered. Consequently, GRP78 is upregulated as a protective response (Ibrahim et al. 2019). In that way, under 25 mM glucose, GRP78 was in control levels (94.04 ± 11.99%) (Fig. 10a), but 50 mM significantly increased GRP78 intracellular expression (124.94 ± 11.11%) (Fig. 10b). Under these treatment conditions, the inhibition of Cyps by CsA significantly reduced GRP78 expression (81.51 ± 9.41%) (Fig. 10b). LPS stimulus also enhanced GRP78 intracellular expression compared to control cells (132.94 ± 1.65%) and 1-h CsA pretreatment significantly decreased GRP78 levels (96.31 ± 8.38%) (Figs. 10a and b). In addition, TG control significantly increased the expression of GRP78 (178.81 ± 24.38%). In summary, the GRP78 response is as expected in an inflammatory response linked to 50 mM glucose levels. In view that HG induces the ER prompting response through the three branches, the presence of calnexin was visualized (Wolf et al. 2024). As Fig. 10c shows, under HG treatment, and mainly under 50 mM glucose, an increase on calnexin presence was detected. Calnexin was increased when cells were treated with LPS. These images reinforce the protein expression results on ER stress.

Fig. 10.

Fig. 10

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Effect of HG in ER response in RAW264.7 cells. Intracellular expression of GRP78 protein under 25 mM (a) and 50 mM (b) glucose. c Confocal images of cells loaded with calnexin and F-actin dyes, Cy3 goat anti-rabbit IgG (red) and Oregon Green (green) respectively. Cells were stimulated with 25 mM (a) and 50 mM (b) glucose for 24 h and pretreated with 1 μM CsA for 1 h, if applicable. LPS at 1 μg/mL was used as positive control of inflammation and TG 1 μM as positive control of ER modulation. Band intensity was normalized by GAPDH. Data are mean ± SEM of three independent replicates performed by duplicate. Data are expressed as percentage of control cells (5.5 mM glucose). Statistical differences determined by One way ANOVA test and Tuckey’s post hoc test. *p < 0.05, **p < 0.01 and ***p < 0.001 compared to control cells. # p < 0.05, ## p < 0.01 and ### p < 0.001 between groups

The modulation of ER related pathways promotes an increased production of ROS. Therefore, the next step was to determine if ER macrophage modulation under HG was increasing ROS levels. Under 25 mM glucose treatment, ROS were in control levels (107.31 ± 3.62%), but 50 mM significantly augmented ROS intracellular content compared to control cells (137.11 ± 8.42%) (Fig. 11a). In these conditions, CsA pretreatment significantly reduced ROS levels (104.04 ± 3.47%). The inflammatory stimulus (LPS) also enhanced ROS content (168.95 ± 11.09%) and Cyps inhibition significantly reduced them (109.14 ± 8.68%). TG control significantly increased intracellular ROS production compared to control cells (119.92 ± 0.69%) (Fig. 11a). Finally, since activated NF-κβ drives the expression of proinflammatory factors, the secretion of IL-6 was analyzed (Mao et al. 2025). As observed in Fig. 11b, IL-6 release was slightly increased by 25 mM (14.91 ± 0.69 pg/mL) and 50 mM glucose (16.01 ± 0.02 pg/mL) compared to control cells (13.44 ± 0.13 pg/mL). In these conditions, Cyps inhibition by CsA significantly reduced the secretion of the proinflammatory cytokine under 50 mM glucose. In addition, LPS stimulus significantly enhanced the release of IL-6 to the cell supernatant (474.41 ± 10.62 pg/mL), as well as the control of TG (314.49 ± 5.74 pg/mL). Under LPS inflammatory conditions, CsA pretreatment significantly reduced IL-6 secretion (311.34 ± 61.52 pg/mL). In summary, ROS and IL-6 production are prominently elevated when cells are incubated with an acute inflammatory stimulus such as LPS. In addition, a prolonged and continuous exposure to HG conditions would lead to an inflammatory response of the same magnitude.

Fig. 11.

Fig. 11

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Inflammatory response under HG conditions in RAW264.7 cells. ROS intracellular levels (a) and IL-6 secretion (b) under HG conditions. Cells were stimulated with 25 mM and 50 mM glucose for 24 h and pretreated with 1 μM CsA for 1 h, if applicable. LPS at 1 μg/mL was used as positive control of inflammation and TG 1 μM as positive control of ER modulation. Data are mean ± SEM of three independent replicates performed by duplicate. Data are expressed as percentage of control cells (5.5 mM glucose) or as cytokine levels (pg/mL). Statistical differences determined by One way ANOVA test and Tuckey’s post hoc test. *p < 0.05, **p < 0.01 and ***p < 0.001 compared to control cells. # p < 0.05, ## p < 0.01 and ### p < 0.001 between groups

Discussion

Diet-related hyperglycemia is characterized by the presence of high blood glucose levels, leading to an inflammatory response, which alters the function of macrophages that become activated (Berbudi et al. 2020). This response triggers the stimulation of many intracellular pathways and the release of several proteins including Cyps (Ramachandran et al. 2012, 2018). In the present work we studied the intracellular and extracellular levels of Cyps in macrophages, in the cell supernatant and inside EVs under HG treatment (Supplementary Fig. 5 in Additional File 2). This study shows the first description of Cyps-containing EVs released by RAW264.7 cells under HG and their role in the modulation of ER and inflammatory-related pathways (Fig. 12). So far, no relationship between Cyps, HG, EVs and ER modulation has been previously established.

Fig. 12.

Fig. 12

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Summary of the intracellular signaling cross-talks studied in this work

In the present work, an increase of CypA expression inside macrophages under HG conditions was observed (Fig. 1). Previously, it was reported that hyperglycemia enhances CypA, since higher levels were found in samples from patients with coronary artery disease associated with type 2 diabetes, in diabetic nephropathy and inside microglia (Gegunde et al. 2023; Chiu et al. 2018; Castedo et al. 2025). The involvement of CypA in the formation of the vascular lesion in type 2 diabetes, and in the increase of lipid uptake and migration of macrophages suggests a role for this immunophilin in the inflammatory response by macrophages underlying HG (Ramachandran et al. 2012, 2018). These results were also obtained under LPS stimulation, as previously reported in microglial cells (Flora et al. 2019). CypB intracellular expression was reduced by HG treatment, while higher CypB levels were detected in metabolic syndrome subjects and inside lymphocytes upon inflammatory conditions (Zhang et al. 2017; Gegunde et al. 2021). Nevertheless, the role of CypB is controversial since it acts as a chemotactic agent promoting monocytes migration but also reduces LPS-induced activation in macrophages (Marcant et al. 2012; Pakula et al. 2007). CypC intracellular expression was increased under HG (Fig. 2), as observed in serum from coronary artery disease patients with diabetes being the highest risk factor of CypC increase (Alfonso et al. 2019). Interestingly, intracellular CypC was detected above its molecular weight (23 kDa). This indicates the presence of a glycosylated form of CypC, which is expected to localize in the ER (Stocki et al. 2014). CypD intracellular levels were unaffected under HG incubation compared to control cells, so it suggests that HG does not affect the opening of the mPTP in these cells.

Differences between Cyps levels depending on the inflammatory stimulus could be due to the activation of distinct intracellular pathways. Since they are related to Cyps, GSK3β and ERK1/2 activation by HG was studied. Under HG treatment, the ratio of pERK/ERK was decreased while LPS stimulation induced the activation of this route (Fig. 5). This could be related to the intracellular CypA and CypB levels since LPS induced the expression of both Cyps while HG enhanced CypA and decreased CypB intracellular expression. Indeed, it has been reported that increased CypA and CypB levels induce the activation of ERK1/2 pathway and that CypA and CypB inhibition reduces the phosphorylation of residues implicated in ERK activation (Kim et al. 2012; Li et al. 2020). Moreover, CsA also reduced ERK activation, which could be related to the decreased intracellular CypB levels by CsA. On the other hand, the different ERK profile observed after LPS and HG treatments could be related to the activation kinetics of this pathway. While the highest ERK activation under HG conditions was reported after 60 min and returns to basal levels within 24 h, LPS induced a sustained ERK activation after 24 h (Cheng et al. 2015; Yuan et al. 2022). GSK3β route was unaffected under both HG and LPS stimulation, since no changes on Ser9 phosphorylation were observed (Fig. 5). This suggests that GSK3β pathway is not involved in the inflammatory response under HG treatment.

ERK1/2 and GSK3β intracellular pathways have been reported to be downregulated by AMPK activation, which is related to glucose metabolism since it increased the translocation of glucose transporters to the membrane (Vahidi Ferdowsi et al. 2021; Yuan et al. 2012; Dai et al. 2020). When pAMPK/AMPK ratio was analyzed, an increase of the phosphorylated form of AMPK under 25 mM and 50 mM glucose was observed (Fig. 6). This is consistent with the reduction observed in the activation of ERK1/2. This could be explained as an inhibitory crosstalk between both kinases previously reported (Behera et al. 2018). Nevertheless, GSK3β was unaffected by AMPK activation under HG. AMPK is an energy sensor activated by several stimuli resulting in an adjustment of AMP/ATP ratio (Schultze et al. 2012). Under HG conditions, ATP content inside macrophages was increased (Fig. 6), which correlates with the activation of AMPK pathway (Carling 2017). In fact, when pAMPK/AMPK was reduced by an acute inflammatory stimulus, ATP levels were lower. Interestingly, inhibition of Cyps by CsA in HG conditions reduced ATP content and AMPK activation, suggesting a relationship between AMPK and Cyps and a potential role of these proteins in the metabolic response under hyperglycemia.

CypA and CypB were detected in the extracellular medium of macrophages under all treatment conditions, CypC was only detected under 50 mM glucose and CypD was absent (Supplementary information). The increase in CypC levels could be related to the decrease in the intracellular CypC observed after 50 mM glucose treatment. Cyps presence was also detected in the extracellular medium of microglial cells under HG (Castedo et al. 2025). In these cells an important vesicular-dependent Cyps release was also described. Therefore, this alternative secretion mechanism for Cyps and their receptor was checked in macrophages (Fig. 4 and supplementary information) since Cyps were previously detected inside EVs under different inflammatory conditions (Castedo et al. 2025; Goetzl et al. 2021a, 2021b; Wu et al. 2021; Pasetto et al. 2021). CypA content inside macrophage-derived vesicles is increased after LPS treatment while decreases under HG, this difference could be related to variations in ERK1/2 activation. This pathway is activated by Rho-associated coiled-coil kinase 2 (ROCK2) activation, which triggers CypA release inside EVs (Cheng et al. 2015; Suzuki et al. 2006). Regarding CypB, its content inside vesicles was only increased by CsA treatment and in combination with HG and LPS as it was previously described in keratinocytes (Fearon et al. 2011). When Cyps A and B receptor was analyzed inside vesicles, an increase of the active CD147 form under HG was detected. According to this, glycosylated CD147 was translocated to the surface membrane expression by HG treatment (25 mM and 50 mM glucose) (Fig. 3). In the same treatment conditions, CsA reduced CD147 expression in the membrane, indicating that HG induces the activation of CD147 receptor through Cyps. Therefore, all these results point to an increase in Cyps and CD147 release through EVs under hyperglycemia conditions in macrophages, no previously described.

Since CypB and CypC are ER-resident immunophilins, the effect of HG stimulation on ER modulation was analyzed. Hyperglycemia triggers the expression of many ER sensors and chaperones, a process known as unfolded protein response (UPR), which is an adaptive response to protect cells (Chen et al. 2023; Wang et al. 2021). When UPR is activated, GRP78 is separated from ER sensors leading to the activation of several ER activation pathways (Lee 2014). pPERK is a sensor that induces the phosphorylation of EIF2⍺ leading to the inhibition of protein translation, increase of GRP78 transcription and ROS production (Chen et al. 2023; Lee 2014). Indeed, it was previously described that HG conditions cause mitochondrial dysfunction and ER activation by stimulating PERK pathway (Cao et al. 2021). When macrophages were treated with 25 mM glucose, pPERK levels were increased but pEIF2⍺ intracellular expression was unaffected. In contrast, 50 mM glucose enhanced pEIF2⍺ expression but pPERK remained in control levels (Fig. 7). Importantly, when Cyps were inhibited by treatment with CsA, the activation of this route in hyperglycemia conditions was significantly decreased. This indicates a role of Cyps reducing the ER prompting response of the PERK-EIF2⍺ axis, which have never been described before. When the other two branches of the ER activating response were analyzed, by measuring pJNK and NF-κβ p65 signaling activity, a similar effect as the PERK pathway was detected. Results showed that both axes were activated by HG and inhibited by CsA (Figs. 8 and 9). In addition, the intracellular expression of GRP78, ROS and IL-6 production were enhanced by 50 mM glucose but not under 25 mM glucose incubation (Figs. 10 and 11). In these conditions, Cyps inhibition also reduced the activation of GRP78 and decreased the associated inflammatory response, pointing again to the role of Cyps in this response, which is sustained along the hole axis. In fact, a previous study reported that CypB inhibition enhances GRP78 expression, reinforcing the participation of Cyps in ER modulation in hyperglycemia (Wang et al. 2016). Taking all together, it seems that 24-h treatment with 25 mM glucose activates the initial steps of the PERK-EIF2⍺ axis and induces a weaker activation of the other two ER branches. In contrast, 50 mM is activating the last part of the PERK-EIF2⍺ route and increasing the ATF6 and IRE1α associated signaling-routes. However, treatment with an acute inflammatory stimulus (LPS) sustained the activation of the three branches for 24 h, which was previously described (Du et al. 2021; Wang et al. 2022).

Conclusions

This work is the first report that establishes a relationship between Cyps, EVs, hyperglycemia, AMPK and ER in macrophages. Results indicate that hyperglycemia has a different impact on Cyps intracellular and extracellular levels, explained by variances in the activation of several pathways. The ER response triggered by HG seems to be related to Cyps since their inhibition reduces the stimulation of ER activation pathways. Results reinforce the idea that Cyps act as key modulators of ER activity and inflammatory pathways, and points to them as potential molecular targets in the pathophysiology of hyperglycemia-related pathologies. Altogether these results reveal a complex role for Cyps and their receptor in the inflammatory response underlying hyperglycemia.

Supplementary Information

10020_2026_1425_MOESM1_ESM.pdf (2.5MB, pdf)

Additional file 1: Original western blots.

10020_2026_1425_MOESM2_ESM.pdf (854.4KB, pdf)

Additional file 2: Supporting information.

Acknowledgements

Noelia Castedo is founded by predoctoral Xunta de Galicia fellowship ED481A_2023. We thank Prof. María José Alonso, for the use of the NTA equipment.

Abbreviations

AMPK

AMP-activated protein kinase

ATF6

Activating Transcription Factor 6

CsA

Cyclosporin A

Cyps

Cyclophilins

CypA

Cyclophilin A

CypB

Cyclophilin B

CypC

Cyclophilin C

CypD

Cyclophilin D

EIF2⍺

Eukaryotic translation initiation factor 2⍺

ER

Endoplasmic Reticulum

ERK1/2

Extracellular-Signal-Regulated Kinase 1/2

EVs

Extracellular Vesicles

FBS

Fetal Bovine Serum

GRPs

Glucose Regulated Proteins

GRP78

Glucose Regulated Protein 78

GSK3β

Glycogen Synthase Kinase-3β

HG

High Glucose

IL-6

Interleukin-6

IRE1α

Endoribonuclease Inositol-Requiring Enzyme 1α

JNK

Jun N-terminal kinase

LPS

Lipopolysaccharide

MTT

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide

mPTP

Mitochondrial transition permeability pore

NF-κβ

Nuclear Factor Kappa-light-chain-enhancer of activated B cells

NTA

Nanoparticle Trafficking Analysis

PBS

Phosphate Buffered Saline

PERK

Protein kinase RNA-like Endoplasmic Reticulum Kinase

PPIase

Peptidyl-Prolyl cis–trans isomerase

PVDF

Polyvinylidene Difluoride

ROCK2

Rho-associated Coiled-Coil Kinase 2

ROS

Reactive Oxygen Species

SDS

Sodium Dodecyl Sulfate

SERCA

Sarco/Endoplasmic Reticulum Ca2+ ATPase

TEM

Transmission Electronic Microscopy

TG

Thapsigargin

UPR

Unfolded Protein Response

Authors’ contributions

N.C.: Investigation, Methodology, Formal analysis, Writing-original draft; R.A.: Conceptualization, Methodology, Funding acquisition, Supervision, Writing-review & editing; A.A.: Conceptualization, Methodology, Funding acquisition, Supervision, Writing-review & editing; M.R.V.: Conceptualization, Supervision, Writing-review & editing; L.M.B.: Conceptualization, Methodology, Funding acquisition, Supervision, Writing-review & editing.

Funding

The research leading to these results has received funding from the following grants. From Conselleria de Cultura, Educacion e Ordenación Universitaria, Xunta de Galicia, GRC (GI-1682–2025). From Ministerio de Ciencia, Innovación y Universidades, PID 2023-149618OB-I00, Grant CPP2021-008447 funded by MCIN/AEI/10.13030/501100011033 and by The European Union NextGenerationEU/PRT. From Interreg EAPA-0032/2022 – BEAP-MAR (cofunded by the EU) and EAPA_0130/2024 – REVALGAE (cofunded by the EU), from European Union HORIZON-CL6-2023-CIRCBIO-01 COMBO—101135438. Noelia Castedo is founded by predoctoral Xunta de Galicia fellowship ED481A_2023.

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Amparo Alfonso, Email: amparo.alfonso@usc.es.

Luis M. Botana, Email: luis.botana@usc.es

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

10020_2026_1425_MOESM1_ESM.pdf (2.5MB, pdf)

Additional file 1: Original western blots.

10020_2026_1425_MOESM2_ESM.pdf (854.4KB, pdf)

Additional file 2: Supporting information.

Data Availability Statement

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Articles from Molecular Medicine are provided here courtesy of The Feinstein Institute for Medical Research at North Shore LIJ

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