Sustained high glucose exposure sensitizes macrophage responses to cytokine stimuli but reduces their phagocytic activity

Abstract

Background: 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.

Results: 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.

Conclusion: Long-term high glucose sensitizes macrophages to cytokine stimulation and reduces phagocytosis and nitric oxide production, which may be related to impaired glycolytic capacity.

Keywords: Cytokine; Diabetes; Glycolysis; Macrophages; Phagocytosis.

Fig. 1

Long-term HighGlu does not affect differentiation, viability or proliferation of BMDMs. Bone marrow cells were cultured for 7 days under NormGlu, HighGlu or Mannitol (osmolarity control) conditions. a Flow cytometric analysis showing the percentage of F4/80⁺ cells differentiated under the three culture conditions. b MTT assay demonstrating the cell viability of BMDMs. c Graph showing the percentage of Ki67⁺ cells. d Representative images of Ki67⁺ cells (red), counterstained with DAPI (blue). e Western blot for Casp3 using protein extracts from the three culture conditions. BMDMs treated with TNFα were used as a positive control. f Representative images of TUNEL assay (red). DNase-treated BMDMs were used as a positive control. Scale bars = 25 μm. Data are represented as mean ± SEM. One-way ANOVA followed by Tukey’s multiple comparison post hoc test was performed

Fig. 2

The effects of short and long-term HighGlu on BMDM gene expression and NO production. Bone marrow cells were cultured for 7 days under NormGlu (white bars) or HighGlu (black bars) conditions. They were then plated and stimulated with LPS + IFNγ (M1) or IL-4 (M2) for 24 h. For short-term HighGlu, cells differentiated under NormGlu, were exposed to HighGlu for 24 h along with M1 or M2 stimulation. a-c qRT-PCR showing the expression of immune-related genes in naïve (a), LPS + IFNγ (b) or IL-4 (c) treated BMDMs after short-term exposure to HighGlu treatment. d Graph showing the concentration of NO in supernatants of naïve and LPS + IFNγ-treated BMDMs under NormGlu or short-term HighGlu conditions. e-g qRT-PCR showing the expression of immune-related genes in naïve (e), LPS + IFNγ (f) or IL-4 (g) treated BMDMs after long-term exposure to HighGlu treatment. h NO concentration in the supernatants of naïve and LPS + IFNγ stimulated BMDMs after long-term HighGlu. Data are represented as mean ± SEM. Unpaired, two-tailed Student’s t test was performed. * p < 0.05, ** p < 0.01, *** p < 0.001

Fig. 3

The effects of short- and long-term HighGlu on IL-1β secretion and LPS/cytokine receptor expression. Bone marrow cells were cultured for 7 days under NormGlu (white bars) or HighGlu (black bars) conditions. They were then plated and stimulated with LPS + IFNγ (M1) for 24 h. For short-term HighGlu, cells differentiated under NormGlu, were exposed to HighGlu for 24 h along with M1 stimulation. a-b ELISA showing the levels of IL-1β in the supernatants of M1 BMDMs exposed to short-term (a) or long-term (b) HighGlu. c-d qRT-PCR showing the expression of LPS and cytokine receptors in naïve BMDMs exposed to short-term (c) or long-term (d) HighGlu. Data are represented as mean ± SEM. Unpaired, two-tailed Student’s t test was performed. * p < 0.05, ** p < 0.01

Fig. 4

Long-term HighGlu and diabetes mellitus affect the phagocytic and bactericidal functions of macrophages. Bone marrow cells were cultured for 7 days under NormGlu, HighGlu or Mannitol conditions. Peritoneal macrophages were isolated from control, diabetic (DM) or ex-diabetic (ex-DM) mice. Phagocytosis was assessed using pHrodo S. aureus bioparticles and bactericidal function using P. aeruginosa PAO1 cultures. The expression of CD16/32, CD36 and CD206 was measured by flow cytometry. a Phagocytic activity of BMDMs cultured under NormGlu (red), HighGlu (green) or Mannitol (blue) conditions. b Phagocytic activity of peritoneal macrophages. c Bactericidal function of BMDMs. d Mean fluorescence intensity of CD16/32, CD36 and CD206 in BMDMs. White bars: NormGlu, black bars: HighGlu, grey bars: Mannitol. Data are represented as mean ± SEM. Two-way ANOVA with bonferroni correction was performed in (a); * p < 0.05, *** p < 0.001 relative to NormGlu, ^♯♯♯ p < 0.001 relative to Mannitol. One-way ANOVA followed by Tukey’s multiple comparison post hoc test was performed in (b-d); * p < 0.05, ** p < 0.01, *** p < 0.001

Fig. 5

HighGlu treatments affect the glycolytic pathway of BMDMs. Bone marrow cells were cultured for 7 days under NormGlu, HighGlu or Mannitol conditions (long-term treatments). For short-term HighGlu and Mannitol, cells differentiated under NormGlu, were exposed to HighGlu or Mannitol for 24 h, respectively. The glycolysis stress assay was performed using the Seahorse XFe96 analyzer. a Representative profile after glycolysis stress assay showing the ECAR of BMDMs exposed to short-term NormGlu (red), HighGlu (green) or Mannitol (blue). b Graph showing non-glycolytic acidification, glycolysis, glycolytic capacity and glycolytic reserve of BMDMs after short-term NormGlu (white bars), HighGlu (black bars) and Mannitol (grey bars) treatments. c Representative profile after glycolysis stress assay showing the ECAR of long-term treated BMDMs. d Graph showing non-glycolytic acidification, glycolysis, glycolytic capacity and glycolytic reserve of BMDMs after long-term exposure to the different conditions. Data are represented as mean ± SEM. One-way ANOVA followed by Tukey’s multiple comparison post hoc test was performed; * p < 0.05, ** p < 0.01, *** p < 0.001

Fig. 6

HighGlu treatments has no effect on the mitochondrial oxidative phosphorylation of BMDMs. Bone marrow cells were cultured for 7 days under NormGlu, HighGlu or Mannitol conditions (long-term treatments). For short-term HighGlu and Mannitol, cells differentiated under NormGlu, were exposed to HighGlu or Mannitol for 24 h, respectively. The Mito stress assay was performed using the seahorse XFe96 Analyzer. a Representative profile after Mito stress assay showing the OCR of BMDMs exposed to short-term NormGlu (red), HighGlu (green) or Mannitol (blue). b Graph showing basal OCR, proton leakage, maximal respiration, spare capacity, non-mitochondrial respiration and ATP production of BMDMs after acute NormGlu (white bars), HighGlu (black bars) and Mannitol (grey bars) treatments. c Representative profile after Mito stress assay showing the OCR of long-term treated BMDMs. d Graph showing the different aspects of mitochondrial oxidative phosphorylation pathway of BMDMs after long-term exposure to the different conditions. Data are represented as mean ± SEM. One-way ANOVA followed by Tukey’s multiple comparison post hoc test was performed

Fig. 7

Schematic illustration showing the experimental setup. BM was collected and cultured under NormGlu, HighGlu or Mannitol conditions for 7 days (long-term treatments). BMDMs were then counted and stimulated with LPS + IFNγ (M1) or IL-4 (M2) for 24 h. BMDMs differentiated under NormGlu were exposed to short-term HighGlu or Mannitol, along with M1 or M2 stimulations for 24 h