Sustained high glucose exposure sensitizes macrophage responses to cytokine stimuli but reduces their phagocytic activity
Macrophages are tissue resident immune cells important for host defence and homeostasis. During diabetes, macrophages and other innate immune cells are known to have a pro-inflammatory phenotype, which is believed to contribute to the pathogenesis of various diabetic complications. However, diabetic patients are highly susceptible to bacterial infections, and often have impaired wound healing. The molecular mechanism underlying the paradox of macrophage function in diabetes is not fully understood. Recent evidence suggests that macrophage functions are governed by metabolic reprograming. Diabetes is a disorder that affects glucose metabolism; dysregulated macrophage function in diabetes may be related to alterations in their metabolic pathways. In this study, we seek to understand the effect of high glucose exposure on macrophage phenotype and functions.
Bone marrow cells were cultured in short or long term high glucose and normal glucose medium; the number and phenotype of bone marrow derived macrophages were not affected by long-term high glucose treatment. Short-term high glucose increased the expression of IL-1β. Long-term high glucose increased the expression of IL-1β and TNFα but reduced the expression of IL-12p40 and nitric oxide production in M1 macrophage. The treatment also increased Arg-1 and IL-10 expression in M2 macrophages. Phagocytosis and bactericidal activity was reduced in long-term high glucose treated macrophages and peritoneal macrophages from diabetic mice. Long-term high glucose treatment reduced macrophage glycolytic capacity and glycolytic reserve without affecting mitochondrial ATP production and oxidative respiration.
Long-term high glucose sensitizes macrophages to cytokine stimulation and reduces phagocytosis and nitric oxide production, which may be related to impaired glycolytic capacity.
KeywordsMacrophages Diabetes Cytokine Phagocytosis Glycolysis
Bone marrow derived macrophages
Extracellular acidification rate
High glucose (30.5 mM)
Normal glucose (5.5 mM)
Oxygen consumption rate
Reactive oxygen species
Macrophages are tissue resident immune cells important for host defence and homeostasis. During inflammation, circulating monocytes are recruited and differentiated into macrophages at the site of tissue damage and infection. The key functions of these macrophages are to eliminate insults or pathogens and to promote tissue repair. During diabetes, macrophages and other innate immune cells are known to have a pro-inflammatory phenotype, which is believed to contribute to the pathogenesis of various diabetic complications, including diabetic nephropathy, atherosclerosis, and retinopathy . Uncontrolled monocyte and neutrophil activation may contribute to diabetes-mediated vascular damage through abnormal leukocyte-endothelial interaction or leukostasis [2, 3]. Inhibition of inflammation has been proposed as a therapeutic approach for diabetic complications [4, 5]. However, diabetic patients are highly susceptible to bacterial infections, and often have impaired wound healing . It is believed that this is partially related to the reduced bactericidal function and wound healing capacity of innate immune cells in diabetic patients. The molecular mechanism underlying the paradox of macrophage function in diabetes is not fully understood.
Recent evidence suggests that macrophage functions are governed by metabolic reprograming . Metabolic pathways not only provide energy in the form of adenosine triphosphate (ATP), but also regulate macrophage functions . For example, during acute inflammation, the classically activated macrophages (M1) predominately utilize glycolysis, but not mitochondrial oxidative phosphorylation, to support their inflammatory function . This metabolic pathway generates amino acids, ribose and nicotinamide adenine dinucleotide phosphate (NADPH) that are necessary for cytokine synthesis, DNA replication and production of reactive oxygen species (ROS) . In addition, glycolytic enzymes and intermediate products are actively involved in the regulation of cytokine production. The pyruvate kinase M2 (PKM2) has been shown to induce IL-1β expression through activating hypoxia inducible factor 1, alpha subunit (HIF1α) , whereas succinate enhances the stabilization of HIF1α and supports IL-1β expression . Since diabetes is a disorder that affects glucose metabolism, we seek to understand whether dysregulated macrophage function in diabetes is related to alterations in their metabolic pathways.
In this study, we investigated the effect of high glucose on bone marrow cell proliferation and differentiation into macrophages. We further investigated the metabolic pathways of macrophages generated under high glucose and normal glucose conditions and how this is related to their functions. We found that sustained high glucose treatment impairs the glycolysis pathway in macrophages, which may be related to their reduced bactericidal activity and ROS production.
The effect of long-term high glucose on bone marrow cell proliferation and differentiation
Ki67 is a nuclear protein necessary for cell proliferation . There was no significant difference in the percentage of Ki67+ cells over the total number of cells (DAPI+) between the different treatment groups (Fig. 1c, d). Western blot did not detect the cleaved form of Caspase-3 (Fig. 1e) in any of the experimental groups. No apoptotic cells were detected in the treatment groups by TUNEL staining (Fig. 1f). Our results suggest that long-term HighGlu treatment did not affect macrophage differentiation from bone marrow cells and did not induce macrophage apoptosis.
Short-term and long-term high glucose treatment on BMDM immune gene expression
BMDMs generated under long-term HighGlu condition had significantly higher levels of IL-1β expression, but the expression of other immune genes such as iNOS, TNFα, IL-6, IL-12p40, Arg-1 and IL-10 was not affected compared with BMDMs generated under NormGlu conditions (Fig. 2e). Following M1 polarization, BMDMs generated under long-term HighGlu conditions expressed lower levels of IL-12p40, but higher levels of TNFα and IL-1β compared to M1 BMDMs under NormGlu conditions (Fig. 2f). Long-term HighGlu treatment also reduced the expression of iNOS, although not significantly (Fig. 2f). In contrast with the iNOS expression, NO production was significantly lower in long-term HighGlu treated M1 macrophages (Fig. 2h). Interestingly, IL4-induced upregulation of Arg-1 and IL-10 was significantly enhanced in long-term HighGlu treated BMDMs (Fig. 2g). Mannitol did not affect the expression levels of the aforementioned cytokines in BMDMs (data not shown). The results suggest that long-term HighGlu may alter macrophage response to cytokine stimulations. In response to LPS + IFNγ stimulation, they have reduced ability to generate NO, but have increased TNFα and IL-1β expression. They also appear to be more sensitive to Th2-type cytokine (e.g., IL-4) stimulation.
Since macrophages generated under NormGlu and HighGlu conditions responded differently to LPS + IFNγ and IL-4 stimulations, we investigated whether they expressed different levels of relevant receptors. Short-term HighGlu only increased Toll-like receptor 4 (TLR-4) expression in BMDMs (Fig. 3c), whereas long-term HighGlu significantly increased the expression of TLR-4, interferon gamma receptor 1 (IFNγR1) and interferon gamma receptor 2 (IFNγR2) (Fig. 3d). Interestingly, the expression levels of interleukin 4 receptor, alpha (IL4ra) remained unchanged in both short-term and long-term HighGlu treated macrophages (Fig. 3c-d).
The effect of long-term high glucose on BMDM phagocytosis and bactericidal function
To further confirm the effect of long-term HighGlu on macrophage phagocytic function, we harvested elicited peritoneal macrophages from STZ-induced diabetic mice, ex-diabetic mice and healthy controls and performed the same phagocytosis assay. Peritoneal macrophages from diabetic mice showed significantly reduced phagocytic capacity when compared to age-matched healthy control mice (Fig. 4b). Interestingly, peritoneal macrophages from ex-diabetic mice had normal levels of phagocytosis, suggesting that hyperglycaemia-induced phagocytosis impairment is reversible.
The pHrodo S. aureus bioparticles assay not only detects the uptake of bacterial particles, but also measures the formation of phagolysosomes, a pivotal action by macrophage to kill bacteria. The bactericidal function of macrophages was further assessed by challenging macrophages with live pathogenic bacteria. After 1 h incubation of P. aeruginosa PAO1 with BMDMs, extracellular as well as intracellular bacteria were quantified. Extracellular bacterial colonies were comparable between the different groups (Fig. 4c), suggesting that the same number of bacteria were taken up by the different macrophages. Interestingly, the colonies of intracellular surviving bacteria in macrophages generated under long-term HighGlu conditions were significantly higher than those in macrophages generated under NormGlu and Mannitol conditions (Fig. 4c). The results suggest that long-term HighGlu impairs bactericidal ability of macrophages.
To understand if the reduced phagocytosis in BMDMs generated under HighGlu conditions seen at 1.5 h and 2 h (Fig. 4a) is related to altered scavenger receptor expression, the expression of cell surface receptors CD16/32, CD36 and CD206 in different types of BMDMs was examined by flow cytometry. Our results show that the expression levels of CD16/CD32 were comparable (Fig. 4d). However, the expression of CD36 and CD206 was significantly lower in BMDMs cultured under HighGlu conditions compared to those cultured under NormGlu or Mannitol conditions (Fig. 4d).
The effect of long-term high glucose on macrophage glucose metabolism and mitochondrial oxygen consumption
Our results suggest that mitochondrial oxygen consumption was not affected by short-term or long-term high glucose treatment. The reduced glycolysis might contribute to high glucose-mediated altered macrophage functions.
In this study, we show that BMDMs generated under long-term high glucose conditions pose a number of intrinsic changes compared to cells generated under normal glucose conditions. Under naïve conditions BMDMs express higher levels of IL-1β and upon LPS + IFNγ stimulation they produce higher levels of TNFα but lower levels of nitric oxide. They also express higher levels of Arg-1 and IL-10 upon IL-4 stimulation compared with BMDMs generated under normal glucose conditions. The phagocytosis and bactericidal activity of these cells appear to be impaired. The phenotype and function of these long-term high glucose treated cells mirror the monocyte/macrophage functions observed in diabetic animals and patients, i.e. they are pro-inflammatory and contribute to the development of diabetic complications, but have reduced defence and tissue repair capacities. This in vitro system is therefore, a good model to study diabetes-mediated immune dysfunction.
Dysregulated innate immune activation is known to contribute to diabetic complications [1, 5, 6, 12]; whether this is due to the intrinsic change of innate immune cells or the noxious tissue microenvironment remains elusive. Results from diabetic animals and patients are controversial. The volume of bone marrow and the hematopoietic fraction were reduced in STZ-induced diabetic mice . The populations of circulating F4/80+, CD11b+, CCR2+ or Ly6G+ cells in diabetic mice were reported to be reduced in some studies , but increased in others . Mononuclear cells from diabetic patients secrete less IL-1 and IL-6 after LPS stimulation [15, 16]. Increased glycation is known to inhibit IL-10 and TNFα production by immune cells , which suggests that the lower cytokine production may be a consequence of diabetes-mediated intrinsic defect in these cells. In our study, long-term HighGlu did not affect the differentiation of bone marrow cells into F4/80+ macrophages, but the function of these cells appears to be altered (see below).
Nitric oxide (NO) is a signalling molecule critically involved in inflammation [18, 19]. Sustained production of NO endows macrophages with cytostatic or cytotoxic activity against viruses, bacteria and other external pathogens . BMDMs cultured under long-term high glucose conditions produced less NO upon LPS + IFNγ stimulation, although the mRNA expression levels of iNOS did not show any significant reduction. It is possible that other isoforms of NOS (e.g., eNOS) or the availability of arginine in long-term high glucose treated macrophages may be affected. Additionally, the long-term high glucose may also affect the NO production at posttranscriptional, translational and posttranslational levels  . They also express lower levels of IL-6 and IL-12p40 and have impaired phagocytic and bactericidal functions. Reduced NO production has been reported in high glucose treated human macrophage J774 cells . Increased NO production  and lower ROS production  has been observed in peritoneal macrophages from diabetic rats. BMDMs and peritoneal macrophages from the NOD mice had reduced ability to phagocytise apoptotic cells [25, 26, 27]. Reddy and colleagues  reported reduced expression of iNOS, IL-10 and Arg-1 in BMDM from db/db mice. All these results suggest that diabetes or long-term high glucose down-regulates iNOS expression and reduces NO production. The molecular mechanism underlying the differential responses to cytokine stimulations between short-term and long-term HighGlu treated macrophages remains elusive. We believe that the long-term HighGlu treated macrophages resemble their in vivo counterparts in diabetic conditions.
Despite reduced NO production following long-term HighGlu treatment, these cells express higher levels of TNFα and IL-1β, suggesting a pro-inflammatory phenotype. This agrees with a previous study, which showed that the myeloid cells recruited to wounds of diabetic mice are intrinsically primed to be more pro-inflammatory than those of non-diabetic mice . TNFα and IL-1β were also higher in cultures of peritoneal macrophages in STZ-induced diabetic rats comparing to control rats . High glucose leads to increased production of IL-1β and TNFα via the NF-κB pathway [30, 31]. Interestingly, the long-term HighGlu treated macrophages also express higher levels of Arg-1 and IL-10 following IL-4 stimulation, suggesting an enhanced M2 phenotype. A previous study by Cucak and colleagues  reported an M2-type phenotype in macrophages from 28-week db/db mice. Our data suggest that long-term high glucose sensitizes macrophages to cytokine stimulations i.e., they present exaggerated responses to both Th1- and Th2-type cytokine stimulation, which may contribute to the dysregulated immune response observed in diabetic patients.
The molecular mechanism underlying long-term HighGlu-mediated alteration of macrophage function remains unknown. Glucose uptake in macrophages is mediated predominately by glucose transporter 1 , which does not rely on insulin signalling. We have found that the HighGlu treatment impairs macrophage glycolytic capacity and glycolytic reserve without affecting mitochondrial ATP production and oxidative respiration. Glycolytic capacity is a measure of the maximum rate of conversion of glucose to pyruvate or lactate by a cell, and the difference between basal glycolysis and glycolytic capacity is known as glycolytic reserve. The basal glycolysis did not differ between BMDMs under NormGlu and HighGlu. Therefore, the reduction in glycolytic reserve may contribute predominately to impaired glycolytic capacity. Glycolytic reserve is an important bioenergy source in response to increases in ATP demand. Our results suggest that long-term HighGlu treatment may limit macrophage energy supply when additional ATP is needed. The LPS-induced proinflammatory macrophages are known to be fuelled by glycolysis . High levels of glycolysis generate NADPH to support NO and ROS production. The reduced glycolytic capacity may explain the lower levels of NO production and impaired bactericidal activity in long-term HighGlu treated macrophages. It may also be related to the reduced phagocytosis of these cells, as we have recently shown that macrophage phagocytic function is directly associated with glycolytic function .
Our study shows that long-term HighGlu sensitizes macrophages to cytokine stimulations, but reduces phagocytosis and bactericidal function of macrophages. The latter may be related to the reduced glycolytic capacity and glycolytic reserve of these cells. Further understanding of the link between altered glucose metabolism and dysregulated macrophage function in diabetes will be important to uncover the immunopathogenesis of diabetic complications.
C57BL/6 J mice were purchased from Harlan and housed under specific pathogen-free conditions with free access to normal chow and water. Diabetes was induced in 12-week male C57BL/6 J mice by intraperitoneal injection of streptozotocin (STZ) at 50 mg/kg for 5 consecutive days . Non-fasting glucose level was measured 1 week after the last injection; mice with glucose level over 13 mmol/l were considered diabetic. If the glucose level was reversed back to normal within 2 months after STZ injection, the mice were considered as ex-diabetic. Body weight was monitored monthly (Additional file 2: Figure S2). Mice were sacrificed 3 months after STZ injection and peritoneal macrophages were harvested from diabetic mice (n = 3), age-matched controls (n = 3) and ex-diabetic mice (n = 3). Mice were euthanized by exposure to CO2. All animal procedures were approved by the UK Home Office under the Home Office Animal Act (1986) (PPL 2773).
Bone marrow-derived macrophage culture
Bone marrow derived macrophages (BMDMs) were cultured from 8 to 12-week-old C57BL/6 J mice using a protocol described previously . Briefly, bone marrow cells were cultured in complete DMEM (Thermo Fisher Scientific, Waltham, Massachusetts, USA) supplemented with 10% heat-inactivated foetal bovine serum (FBS) and 20% L929-conditioned media as a source of macrophage colony-stimulating factor (M-CSF) . The phenotype of BMDMs was confirmed by flow cytometry as being F4/80+ and CD11b+ (data not shown).
High glucose treatment of BMDMs
BMDMs were generated from bone marrow cells under NormGlu condition. The cells were collected on day 7 and plated in 12- or 96-well plates with NormGlu, HighGlu or Mannitol for further investigations.
BMDMs were generated under NormGlu, HighGlu or Mannitol conditions. On day 7, cells were harvested and plated in 12- or 96-well plates for further investigations.
Macrophage polarization with cytokines
BMDMs were seeded in 12-well plates at a density of 5 × 105 cells per well. The cells were polarized into M1 with 50 ng/ml LPS (Sigma-Aldrich, St. Louise, Missouri, USA) + 100 ng/ml IFNγ (Bio-Techne, Minneapolis, Minnesota, USA) or M2 with 20 ng/ml IL-4. (Bio-Techne, Minneapolis, Minnesota, USA) The supernatants were collected 24 h after cytokine stimulation and nitric oxide (NO) was measured using the Griess Reagent System (Promega, Madison, Wisconsin, USA), according to manufacturer’s instructions. IL-1β in BMDM supernatants was measured using the mouse IL-1 beta/IL-1F2 DuoSet ELISA kit (DY401, Bio-Techne, Minneapolis, Minnesota, USA) according to manufacturer’s instructions. Cells were collected for flow cytometry, protein or RNA extraction.
Assessment of BMDMs cell death and proliferation
Cell death of BMDMs cultured under different glucose conditions (NormGlu, HighGlu or Mannitol control) were measured using Thiazolyl Blue Tetrazolium Blue (MTT, Sigma-Aldrich, St. Louise, Missouri, USA)  and TUNEL assay (In Situ Cell Death Detection Kit, TMR red, Roche, Basel, Switzerland), according to manufacturer’s instructions. Cell proliferation was assessed by Ki67 (1:100, clone: SolA15, Thermo Fisher Scientific) immunofluorescence, using a standard protocol . For these assays, cells were counted on the day of bone marrow extraction and 1 × 104 cells were plated in a 96-well plate. After 7 days of differentiation the assays were performed.
For MTT assay, cells were incubated in 0.5 mg/ml MTT for 4 h at 37 °C. The MTT solution was then replaced by 200 μl DMSO per well and cells were incubated for 30 min at 37 °C until the purple formazan crystals were dissolved. Absorbance was measured at 562 nm using the Fluostar Omega plate reader (BMG Labtech, Ortenberg, Germany). For the TUNEL assay, cells were fixed in 2% PFA for 15 min at room temperature. After several washes with PBS, cells were incubated with the TUNEL-mix for 1 h at 37 °C. After washes with PBS, cells were imaged using an IX51 inverted fluorescent microscope (Olympus, Southend, UK). Cells were treated with DNase (Thermo Fisher Scientific) for 10 min at room temperature, before the addition of the TUNEL-mix, as a positive control.
Total protein was extracted in RIPA buffer supplemented with 10% proteinase inhibitor cocktail (Sigma-Aldrich) and quantified using the BCA assay (Thermo Fisher Scientific). The same amount of denatured proteins from each treatment group was separated on a 12% SDS-PAGE gel and transferred onto polyvinylidene fluoride (PVDF) membranes. Membranes were incubated with antibody against mouse Caspase-3 (1:100, Millipore, Darmstadt, Germany) at 4 °C overnight, followed by anti-rabbit-HRP (1:5000, Agilent Technologies, Santa Clara, California, USA) for 1 h at room temperature. Protein from BMDMs stimulated with 20 ng/ml TNFα for 3 h at 37 °C was used as a positive control for the cleaved Caspase-3. Membranes were developed using enhanced chemiluminescence (Clarity™ Western ECL Blotting Substrates, BioRad, Hercules, California, USA) and corresponding bands were detected using Syngene G-Box imaging system (Syngene, Cambridge, UK).
Quantitative real time RT-PCR
Primers used for RT-PCR
Flow cytometric analysis
Single-cell suspensions were stained for cell surface markers using specific antibodies following protocols published previously . All samples were analysed using a FACS Canto II (BD biosciences, UK) and data were processed using FlowJo Software (version 7 for Windows, Tree Star, Ashland, OR, USA). The following antibodies were used: F4/80 (clone: BM8, Thermo Fisher Scientific), CD16/32 (clone: 2.4G2, Thermo Fisher Scientific); CD36 (clone: HM36, Biolegend, San Diego, California, USA), CD206 (clone: C068C2, Biolegend).
Phagocytosis was conducted using the pHrodo S. aureus bioparticles conjugated phagocytosis kit (Thermo Fisher Scientific) following the manufacturer’s instructions . Briefly, BMDMs or peritoneal macrophages were seeded onto 96-well black-walled plates at a density of 5 × 104 per well for 24 h before adding the bioparticles. Culture medium with bioparticles only was served as a negative control. Fluorescence intensity was measured 1 h, 1.5 h and 2 h after the addition of bioparticles using the Fluostar Omega plate reader (BMG Labtech). The net fluorescence intensity was the difference between the fluorescence intensity from the experimental wells and that from the negative control wells .
Bacterial killing assays
A colony of Pseudomonas aeruginosa PAO1 was selected from a cetrimide agar plate and inoculated into 10 mL of LB broth (Sigma-Aldrich). This was grown overnight at 37 °C in an orbital shaker at 160 rpm. The cells were harvested by centrifugation in an Eppendorf centrifuge 5810R at 4000 rpm for 10 mins and resuspended in 40 ml of fresh LB broth. They were grown to log phase for 4–6 h at 37 °C on a rotary shaker at 160 rpm. The optical density of the bacterial culture was measured at 600 nm (WPA biowave).
To assess their bactericidal activity, 1 × 106 BMDMs were plated in 12-well plates. P. aeruginosa was added to the wells at multiplicity of infection (MOI) 1:10 (1 × 107 CFU/ml). The BMDM/P. aeruginosa co-culture was incubated for 1 h at 37 °C. The coculture was then centrifuged at 1500 rpm for 5 min and the supernatant removed. The supernatant was serial diluted in PBS at 10− 4, 10− 5, 10− 6 and 10− 7 and plated onto cetrimide agar (Sigma-Aldrich). Saponin (Sigma-Aldrich) at 1:1000 dilution in PBS was added to all pellets and incubated for 5 min. The digested pellets were serial diluted in PBS at 10− 4, 10− 5, 10− 6 and 10− 7 and plated onto cetrimide agar. All cetrimide plates were incubated for 18 h and CFU/mL were then calculated.
Bioenergetic profile of BMDMs
BMDMs metabolic profiles were assessed using Seahorse XF Glycolysis Stress Test kit and Cell Mito Stress Test kit with a Seahorse XFe96 Analyzer (Agilent Technologies) following manufacturer’s instructions. Glycolytic activities were assessed by measuring the Extracellular Acidification Rates (ECAR) and mitochondrial activities were assessed by measuring the Oxygen Consumption Rates (OCR). Data were normalised with the total cell number and expressed as mpH/min/104 cells for ECAR or pmol/min/104 cell for OCR.
Data were presented as mean ± standard error (SEM). Unpaired, two-tailed Student’s t test was used for two-group comparisons. One-way ANOVA was used to compare the means of multiple groups; Tukey’s multiple comparisons were used for post hoc test when samples had equal variances. Student t test with Welch’s correction was employed when the equal variance assumption was not met. P < 0.05 was considered statistically significant.
We thank Kevin Harkin for proof reading and providing helpful comments on the manuscript.
This work was supported by Fight for Sight UK (1574/75), Diabetes UK (16/0005537).
Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
SP and JL performed experiments. RI contributed to the study design. HX contributed to the study design and data interpretation. MC oversaw all aspects of the study, including performing experiments, experiment design, data analysis and interpretation and writing of the manuscript. All authors read and approved the final manuscript.
Animal procedures (PPL 2773) were approved by the UK Home Office under the Home Office Animal Act (1986).
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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