We also assessed Nitric Oxide and IL levels, but no significant differences were observed Supplementary Figures 1 and 2. Figure 6 IL-1β secretion by ND-BMDM and D-BMDM with Nigericin priming and LPS stimulation. IL-1β release after A 30 minutes with different glucose concentrations.

IL-1β measurement was performed by enzyme-linked immunosorbent assay. It is already described that patients can establish a chronic inflammation state under diabetic conditions, characterized by a decompensated secretion of pro-inflammatory cytokines, such as TNF-α, IL-1β and IL-6, and it is suggested as the major cause of comorbidities related to diabetes Furthermore, it has been identified that peritoneal macrophages, when exposed to high levels of glucose secrete greater amounts of TNF-α, IL-1β, IL-6 and IL in response to high glucose concentrations 34 , It has also been reported by Cheng et al.

A study showed that, when cultivated in a hyperglycemic medium, BMDM secreted a more significant amount of TNF-α, but the expression of IL-6 under the same conditions was reduced However, our studies observed that high glucose levels alone were not enough to stimulate the secretion of TNF-α, IL-1β and IL-6 by macrophages.

On the other hand, when stimulated with LPS, there was a significant increase in the secretion of these cytokines.

Furthermore, Tessaro et al. Our findings corroborate with previous studies where it was reported that high glucose concentrations 15mM, 25mM do not alter the expression of TNF-α and IL-6 by macrophages, however, when stimulated with LPS, the secretion of these cytokines increase, showing that hyperglycemia it is not a sufficient stimulus for the high production of these cytokines by macrophages 35 , High glucose conditions can cause mitochondrial dysfunction, increase the production of ROS and activates the autophagy pathway 29 , 39 — Furthermore, LC3b protein is involved in mitophagy 42 , a process that removes dysfunctional mitochondria by the autophagic machinery Therefore, we suggest that the high levels of LC3b and beclin-1 expression by ND-BMDM, when compared to D-BMDM identified in this study are due to the normal cellular regulation process in response to stress caused by the hyperglycemic condition, suggesting that the autophagy pathway is impaired in macrophages from diabetic animals, which reinforces that these cells are sensitized when exposed to the hyperglycemic state in vivo 44 — Furthermore, we observed a reduction in LC3b expression by ND-BMDMs and D-BMDMs stimulated and in hyperglycemic conditions.

Our findings corroborate with study that showed a decrease in LC3b expression in THPderived macrophages exposed to high concentrations of glucose with the inflammasome pathway activated However, in this study performed by Dai et al.

It was already described that alterations in the autophagy pathway can directly interfere in the inflammatory response of diabetic individuals, making them susceptible to the development of infections 12 49 , Combined with that, nigericin is known to be an inducer of the NLRP3 inflammasome pathway, where the formation of this complex results in the production of IL-1β In our studies, we observed that macrophages from diabetic animals, when stimulated, secreted a greater amount of the cytokine IL-1β, showing that there is an exacerbated production of this cytokine when stimulated by LPS.

Since the relationship between the autophagy process and IL-1β cytokine secretion has been widely studied, it has been reported that this process is responsible for sequestering this cytokine and preventing its secretion 52 , and a negative regulation of this pathway can lead to an increase of IL-1β release 21 , These results suggest that, besides macrophages of diabetic animals being previously sensitized by hyperglycemia, the failure of the autophagy machinery may be contributing to the decompensated secretion of this cytokine.

With the changes observed in our study, we can observe that hyperglycemia plays an essential role in the inflammatory response of BMDMs from diabetic mice, since the high concentration of glucose with LPS stimulation led to significant changes in the secretion of inflammatory mediators and in the autophagy process, having a direct effect on cellular homeostasis.

The high concentration of glucose alters the inflammatory pathways in macrophages after LPS stimulation, disrupting the secretion of pro-inflammatory cytokines by these cells, leading to an impaired inflammatory response against infections.

Furthermore, we observed that the sensitization caused by hyperglycemia in macrophages can downregulate the expression of proteins involved in the autophagy pathway, impairing cellular homeostasis, suggesting the main role of this mechanism over macrophages under diabetic conditions.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. ES and JM conceived and designed the experiments.

ES, LQ, JG, KP and RB performed the experiments. ES, LQ, SE and JM analyzed the data. ES and JM wrote the paper with the assistance and contribution of all the authors. All authors contributed to the article and approved the submitted version. The authors would like to thank Silene Migliorini and Fabiana Teixeira for providing the acquisition, organization of reagents used in this project and assistance at the laboratory.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers.

Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Watanabe S, Alexander M, Misharin AV, Budinger GRS. The role of macrophages in the resolution of inflammation.

J Clin Invest 7 — doi: PubMed Abstract CrossRef Full Text Google Scholar. Wang Y, Smith W, Hao D, He B, Kong L. M1 and M2 macrophage polarization and potentially therapeutic naturally occurring compounds.

Int Immunopharmacol — Thapa B, Lee K. Metabolic influence on macrophage polarization and pathogenesis. BMB Rep 52 6 — Martin KE, García AJ. Macrophage phenotypes in tissue repair and the foreign body response: Implications for biomaterial-based regenerative medicine strategies.

Acta Biomater — Locati M, Curtale G, Mantovani A. Diversity, mechanisms, and significance of macrophage plasticity. Annu Rev Pathol — Germic N, Frangez Z, Yousefi S, Simon HU. Regulation of the innate immune system by autophagy: monocytes, macrophages, dendritic cells and antigen presentation.

Cell Death Differ 26 4 — Jung S, Jeong H, Yu SW. Autophagy as a decisive process for cell death. Exp Mol Med 52 6 — Wang Y, Li YB, Yin JJ, Wang Y, Zhu LB, Xie GY, et al.

Autophagy regulates inflammation following oxidative injury in diabetes. Autophagy 9 3 —7. Klionsky DJ. Stepping back from the guidelines: Where do we stand? Autophagy 12 2 —4. Jiang GM, Tan Y, Wang H, Peng L, Chen HT, Meng XJ, et al. The relationship between autophagy and the immune system and its applications for tumor immunotherapy.

Mol Cancer 18 1 Deretic V, Kimura T, Timmins G, Moseley P, Chauhan S, Mandell M. Immunologic manifestations of autophagy. J Clin Invest 1 — Cadwell K. Crosstalk between autophagy and inflammatory signalling pathways: balancing defence and homeostasis.

Nat Rev Immunol 16 11 — Deretic V, Levine B. Autophagy balances inflammation in innate immunity. Autophagy 14 2 — Yin X, Xin H, Mao S, Wu G, Guo L. The role of autophagy in sepsis: Protection and injury to organs. Front Physiol Lopez-Castejon G, Brough D.

Understanding the mechanism of IL-1β secretion. Cytokine Growth Factor Rev 22 4 — Kaushik V, Yakisich JS, Kumar A, Azad N, Iyer AKV. Ionophores: Potential use as anticancer drugs and chemosensitizers.

Cancers Basel 10 10 Gao G, Liu F, Xu Z, Wan D, Han Y, Kuang Y, et al. Evidence of nigericin as a potential therapeutic candidate for cancers: A review. BioMed Pharmacother Wang RC, Wei Y, An Z, Zou Z, Xiao G, Bhagat G, et al. Akt-mediated regulation of autophagy and tumorigenesis through beclin 1 phosphorylation.

Science —9. Yoshii SR, Mizushima N. Monitoring and measuring autophagy. Int J Mol Sci 18 9 Qiu P, Liu Y, Zhang J. Review: the role and mechanisms of macrophage autophagy in sepsis.

Inflammation 42 1 :6— Biasizzo M, Kopitar-Jerala N. Interplay between NLRP3 inflammasome and autophagy. Front Immunol Wynn TA, Vannella KM.

Macrophages in tissue repair, regeneration, and fibrosis. Immunity 44 3 — Kaneko N, Kurata M, Yamamoto T, Morikawa S, Masumoto J. The role of interleukin-1 in general pathology. Inflammation Regen CrossRef Full Text Google Scholar. Xiu F, Stanojcic M, Diao L, Jeschke MG.

Stress hyperglycemia, insulin treatment, and innate immune cells. Int J Endocrinol Pavlou S, Lindsay J, Ingram R, Xu H, Chen M. Sustained high glucose exposure sensitizes macrophage responses to cytokine stimuli but reduces their phagocytic activity.

BMC Immunol 19 1 Edgar L, Akbar N, Braithwaite AT, Krausgruber T, Gallart-Ayala H, Bailey J, et al. Hyperglycemia induces trained immunity in macrophages and their precursors and promotes atherosclerosis. Circulation 12 — Fiorentino TV, Prioletta A, Zuo P, Folli F.

Hyperglycemia-induced oxidative stress and its role in diabetes mellitus related cardiovascular diseases. Curr Pharm Des 19 32 — Yan LJ. Pathogenesis of chronic hyperglycemia: from reductive stress to oxidative stress. J Diabetes Res Ighodaro OM. Molecular pathways associated with oxidative stress in diabetes mellitus.

BioMed Pharmacother — Ayala TS, Tessaro FHG, Jannuzzi GP, Bella LM, Ferreira KS, Martins JO. High glucose environments interfere with bone marrow-derived macrophage inflammatory mediator release, the TLR4 pathway and glucose metabolism.

Sci Rep 9 1 Marim FM, Silveira TN, Lima DS Jr, Zamboni DS. A method for generation of bone marrow-derived macrophages from cryopreserved mouse bone marrow cells.

PloS One 5 12 :e Carlos D, Costa FR, Pereira CA, Rocha FA, Yaochite JN, Oliveira GG, et al. Mitochondrial DNA activates the NLRP3 inflammasome and predisposes to type 1 diabetes in murine model. Ramalho T, Filgueiras L, Silva-Jr IA, Pessoa AFM, Jancar S.

Impaired wound healing in type 1 diabetes is dependent on 5-lipoxygenase products. Sci Rep 8 1 Pan Y, Wang Y, Cai L, Cai Y, Hu J, Yu C, et al.

Inhibition of high glucose-induced inflammatory response and macrophage infiltration by a novel curcumin derivative prevents renal injury in diabetic rats.

The results were analyzed using the absolute quantification with arbitrary values. For this purpose, a standard curve was carried out for each target and reference gene.

Primer efficiencies were calculated in each experiment from the standard curve carried out in the same plate as the quantified samples. This calibrator point was the cDNA obtained from the control samples No activated macrophages cultured in normoxia and normoglycemia.

Arbitrary values were then calculated for each condition by comparison with the value 1, using the ratios. Results are presented as mean ± SD standard deviation.

Statistical significance was assessed using one way analysis of variance ANOVA followed by Mann Witney compare all pairs of groups posthoc test.

Prism Excel Stat software was used for all data analysis. The microarray revealed that genes were statistically up or downregulated in hyperglycemia. Filtering for gene expression modulated at least 2 fold, only 54 genes were found to be upregulated and 94 downregulated in hyperglycemia.

Among these genes, thirteen proinflammatory cytokines and ten chemokines were found to be upregulated Fig 2. In contrast, TGF-β1, a crucial cytokine promoting wound healing was downregulated in hyperglycemia 2·01 fold lower.

Moreover, CD36 and Scavenger receptor B, two genes involved in the process of phagocytosis, were also downregulated Table 1. After analysis, the microarray revealed that three major signalling pathways were modulated in hyperglycemia.

These pathways were the EGF Epidermal Growth Factor and Wnt5A Wingless-type MMTV integration site family, member 5A and NOD signalling pathways Table 1. Eleven genes encoding for proteins with an anti-apoptotic effect were also upregulated and three pro-apoptotic genes were downregulated Fig 2.

Surprisingly, no genes encoding for metalloproteinases or other proteases were upregulated when cells were cultivated in hyperglycemia.

Finally, only a few genes involved in glycolysis were modulated by the high glucose concentration. Only two were slightly downregulated for glycolysis and two for proliferation.

Overview of genes upregulated and dowregulated in a high glucose environment and hypoxia. The impact of hypoxia and hyperglycemia on the gene expression of TNF- α, IL-1a, IL-6 and GM-CSF was analyzed in detail and compared to the results obtained with the microarray.

Gene expression was measured 1 hour and 17 hours after macrophage activation by LPS. This activation triggered an increase of the TNF-α gene expression after one hour regardless of the culture conditions. The gene expression was around four times the basal level but was not significantly different whether the cells were cultivated in hypoxia or in hyperglycemia Fig 3A.

As a result, the combination of hypoxia and hyperglycemia has a synergistic effect for long term TNF-α gene expression. A TNF-α, B IL-1a, C IL-6, D CSF2, GM-CSF.

Unlike TNF-α, the LPS activation did not increase the IL-1 gene expression after one hour when cells were cultivated in normoxia. In sharp contrast, hypoxia had a huge effect as the IL-1a gene expression was circa five fold higher for cells cultivated in hyperglycemia and normoglycemia.

After 17 hours, the IL-1a expression decreased to its basal level in normoglycemia whereas that measured in hyperglycemic conditions remained high Fig 3B. After 17 hours post-activation, hyperglycemia and hypoxia are required to maintain a high expression of IL-1A.

The addition of lipopolysaccharide to macrophages triggered a slight increase of IL-6 gene expression irrespective of the culture condition Fig 3C. When hypoxia was combined with hyperglycemic conditions, IL-6 gene expression increased dramatically for this group after 17 hours post activation.

Interestingly, cells cultivated in normoxia and hyperglycemia exhibited an increased expression of IL-6 after 17 hours compared to that after 1 hour Fig 3C.

In this case, hyperglycemia has an effect on its own but it was amplified by hypoxia. Macrophage activation did not have any effect on GM-CSF gene expression of each group one hour after LPS addition Fig 3D.

A long term effect of hypoxia was observed as the gene expression of cells cultivated in hypoxia was half that of those cultivated in normoxia.

Therefore, hyperglycemia and hypoxia have a negative effect on inflammation as they upregulate the gene expression of the inflammatory cytokines TNF-α, IL-6 and IL-1 in activated macrophages. The LPS activation of macrophages led to a slight increase of the CD gene expression in normoxia and normoglycemia Fig 4A.

Hypoxia and hyperglycemia had a long term effect on this gene expression. Low oxygen tension and high glucose concentration negatively impacted the CD expression on their own.

Macrophages exhibited a drastic upregulation of the Class B Scavenger receptor gene when the cells were cultivated in hypoxia. No effect was observed in normal O 2 condition Fig 4B. After 17 hours post-activation, Class B Scavenger gene expression recovered its basal level irrespective of the culture conditions.

However hypoxia had a slight positive effect when cells were cultured in hyperglycemia. Hence, hypoxia and hyperglycemia decreases the abilities of activated macrophages for phagocytosis because the expression of CD and Class B scavenger are downregulated. The TGF-β1 gene expression was not modified one hour after activation of macrophages regardless of the O 2 and glucose conditions Fig 5.

This expression did not change after 17 hours post activation either. Hence a low O 2 tension and hyperglycemic conditions does not have any impact on TGF-β production Fig 5. SOCS-3 was upregulated one hour after LPS activation when the cells were cultivated in hypoxia. This upregulation was higher for macrophages cultivated in normoglycemia Fig 6.

The SOC-3 gene expression decreased to its basal level after 17 hours in these groups. In contrast, the cells cultivated in normoxia and hyperglycemia exhibited an upregulation of SOCS The goal of this study was to analyze the impact of hyperglycemia on the macrophage phenotype focusing on proteins involved in inflammation, proliferation, apoptosis, ECM breakdown and wound healing.

For this purpose, a gene expression microarray analysis was performed on activated macrophages cultured in a hyperglycemic and hypoxic environment with a low quantity of bovine serum with the aim of mimicking the chronic wound milieu.

Subsequently, the effect of hyperglycemia and hypoxia were analyzed separately to understand their contribution in the chronic wounds. Lastly a potential synergistic effect of high glucose concentration and low O 2 tension was evaluated.

Hyperglycemia has several detrimental effects on human homeostasis. A chronic high glucose concentration leads to a process of protein glycation and the production of advanced glycation endproducts AGEs.

AGEs promote macrophage activation via NF- κ B and stimulate the production of reactive oxygen species ROS [ 22 ]. As a consequence, diabetes predisposes to epigenetic changes which lead to chronic inflammation [ 23 ].

The microarray results show that 13 pro-inflammatory cytokines and 10 chemokines were upregulated in hyperglycemia, thereby confirming the perpetual dysregulation of the inflammatory homeostasis. Pro-inflammatory macrophages are more metabolically active in hyperglycemic conditions and exclusively use glucose as a source of energy [ 24 ].

Hence, this mode of energy production can contribute to the failure to resolve inflammation. Chronic wounds are characterized by the recruitment and the persistence of immune cells in the wound bed neutrophils and macrophages [ 25 ]. The results showed the upregulation of 11 anti-apoptotic genes and the downregulation of 3 pro-apoptotic genes, indicating the direct impact of hyperglycemia on the large number of macrophages inside the cutaneous wound bed.

One major feature of impaired wound healing is the massive breakdown of extracellular matrix. High glucose concentration triggers the production and secretion of metalloproteinases such as MMP-9 and MMP-2 by fibroblasts, keratinocytes and macrophages [ 25 , 26 ].

In our conditions, hyperglycemia did not have a direct effect on proteases as only MMP-7 was affected. In addition, this enzyme was slightly downregulated. Lipopolysacharide LPS is an outer membrane component of Gram negative bacteria which activates macrophages [ 27 ].

LPS contact with TLR receptors orientates macrophages towards a pro-inflammatory M1 phenotype. This phenotype is characterized by the production of inflammatory cytokines such as IL-6, IL-1, TNF-α, reactive species of oxygen ROS and NO [ 28 ]. The expression of inflammatory cytokines is based on the NF- κ B activation in macrophages [ 29 ].

AGEs interacting with RAGE, their membrane receptor, can be a continuous activator of NF- κ B. As a result, AGEs increase the production of pro-inflammatory cytokines as previously described [ 30 ].

Hypoxia is associated with the activation of hypoxia inducible factors HIFs which is the key mediator of the induction of IL-6, IL-1, TNF-α [ 31 ]. Hence, hypoxia and hyperglycemia could have a synergistic effect on the production of pro-inflammatory cytokines.

In addition, a cross-talk exists between HIF and NF- κ B to increase this production. We analyzed in detail the impact of hypoxia and high glucose on cytokine production with a kinetic view.

After one hour post LPS activation, the combination of hypoxia and hyperglycemia had a dramatic effect on the expression of TNF-α and IL The combination of hyperglycemia and hypoxia is required to induce a sustained production of pro-inflammatory cytokines as the same phenomenon was observed for TNF-α and IL Beside its major role in inflammation, it has been recently shown that IL-6 could have anti-inflammatory effects via modulation of macrophage phenotype [ 32 ].

IL-6 promote the M2 phenotype of macrophages by inducing the expression of the IL-4 receptor [ 32 ]. In this study, the IL-4 receptor was not upregulated. Several studies have reported on the anti-inflammatory effect of IL-6 and the dependency on the concentration.

In this study, Il-6 was dramatically upregulated and orientated its action towards chronic inflammation [ 32 ]. Regarding IL-1, only hypoxia had a short term impact on the expression of this cytokine. An effect was observable 17 hours post activation for the cells cultivated in hypoxia and hyperglycemia.

This shows their importance for a long term effect on inflammation. Moreover, the sustained and prolonged production of IL-1 contributes to diminish wound healing by activating TLR receptors and maintaining macrophages in a M1 phenotype [ 33 ].

Granulocyte macrophage colony-stimulating factor GM-CSF is highly upregulated in hyperglycemic conditions. GM-CSF is produced during the inflammation phase and is a marker of M1 macrophages [ 34 ]. This cytokine stimulates the production of chemokines such as CCL2 and CCL3 and is involved in the recruitment of myeloid cells within the wound [ 33 ].

The GM-CSF expression is induced by pro-inflammatory cytokines such as IL-1 and TNF-alpha. As a consequence, the high production of pro-inflammatory cytokines by high glucose and low O2 tension increases the expression of GM-CSF, which has also a negative effect on inflammation.

In our conditions, GM-CSF was not impacted by hyperglycemia which is not consistent with the results of the micro array. Suppressor of cytokine signaling 3 SOCS3 is associated with the pro-inflammatory M1 phenotype of macrophages. In addition, SOCS3 decreases the phagocytic activities of macrophages for apoptotic neutrophils.

The decrease of clearance of dead neutrophils impedes the resolution of inflammation and a pro-inflammatory environment shows a strong upregulation of SOCS3 [ 35 , 36 ]. Hyperglycemia seems to have a short term negative effect on SOCS3.

Surprisingly, hyperglycemia seems to favour the resolution of inflammation at this time point. However, hyperglycemia has a negative effect after 17 hours when the cells are cultivated in hyperglycemia.

As SOCS-3 is upregulated in this study, this confirms the inflammatory effect of IL-6 in hyperglycemia. It has been shown this cytokine has an anti-inflammatory effect only when SOCS-3 was downregulated or ablated [ 37 ].

Hyperglycemia combined with hypoxia also led to the upregulation of a panel of chemokines. Among them, CCL-4 is of great interest because it activates neutrophils which can trigger neutrophilic inflammation [ 38 , 39 ].

In addition, this chemokine triggers the production of pro-inflammatory cytokines. Five C-X-C chemokines CXCL 1- CXCL5 were also upregulated in hyperglycemia.

For example, CXCL2 is highly expressed. Moreover, CXCL2 recruit neutrophils to infection sites. Overall, the other chemokines have the same effect, recruiting leucocytes in the wound.

Hence, hyperglycemia and hypoxia create a vicious circle which maintains a high inflammation in the wound and prevents the switch from the inflammatory phase to the proliferative one.

Phagocytosis of dead cells is required for the resolution of inflammation and the transition towards the proliferative phase [ 41 ] because impaired cell clearance has been observed in diabetic wounds [ 42 ]. CD36 is a member of the class B scavenger receptor family found in macrophages. CD36 is an efferocytosis receptor which acts in combination with α v β 3 integrin to engulf dead neutrophils [ 41 ].

Unlike the normoglycemic conditions, CD36 expression does not increase in hyperglycemia one hour after LPS activation. This result shows the impaired phagocytic activities of macrophages cultivated in high glucose. In addition, CD36 mediate s the bacteria phagocytosis and the production of inflammatory molecules such as IL-8 [ 43 ].

Hence, the absence of an upregulation of CD36 following the activation by LPS suggests the lower ability of macrophages to combat infection when they are in a hyperglycemic milieu.

Class B scavenger type I receptors CLA-1 are also involved in the pathogen s recognition and the removal of apoptotic cells.

They have a lot of structural similarities with CD36 [ 43 ]. They also have an effect on cytokine production as Knock Out CLA-1 mice expressed more inflammatory cytokines than the wild type [ 43 ]. The results showed that hypoxia is an important stimulus for Class B scavenger expression because its expression is multiplied by 12 in hypoxia over that in the normoxic conditions.

Hyperglycemia negatively modulates this upregulation showing once again the impaired phagocytic abilities of diabetic macrophages, thereby settling down the chronic inflammation in the cutaneous wound. TGF-B1 is a master regulator of the wound healing process by promoting the switch between the inflammation and the proliferative phase [ 44 ].

The TGF-B activity counterbalances the effect of TNF-alpha in macrophages [ 45 ] and favours angiogenesis, ECM deposition and fibroblast proliferation. Hyperglycemia and hypoxia did not have any effect on its gene expression.

Hence, hyperglycemia only negatively impacts the expression of pro-inflammatory cytokines but not those involved in wound healing. Hyperglycemia has a negative impact on the wound healing of foot diabetic ulcers.

High glucose level acts in synergy with hypoxia to maintain the state of chronic inflammation observed in chronic wounds. Hyperglycemia increases the expression of pro-inflammatory cytokines and chemokines by macrophages and decreases their ability of phagocytosis, required for the resolution of inflammation.

By contrast, the cytokines involved in wound healing were not impacted by the high glucose concentration. This overview of the macrophage behavior cultivated in hyperglycemia and hypoxia could be helpful towards discovering novel relevant targets for the treatment of foot diabetic ulcers.

The authors would like to thank Dr Oliver Carroll for his technical guidance in the project, Dana Toncu for editorial and critical assessment of the manuscript, and Mr Anthony Sloan for his editorial assistance in finalizing the manuscript.

Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Article Authors Metrics Comments Media Coverage Reader Comments Figures. Abstract Diabetic foot ulcers DFUs are characterized by a chronic inflammation state which prevents cutaneous wound healing, and DFUs eventually lead to infection and leg amputation.

Introduction Diabetic foot ulcers are the most common, painful and crippling complications of diabetes mellitus [ 1 ]. In pathological conditions, macrophages are locked in the M1 phenotype, thereby leading to chronic inflammation Hypoxia in DFU creates conditions that are disadvantageous because the low oxygen tension induces the increased release of pro-inflammatory cytokines via the activation of NF- κ B signaling pathways [ 10 , 11 ].

Download: PPT. Fig 1. Differentiation and activation of macrophages cultivated in hyperglycemia and hypoxia. Results 3. Table 1.

Gene expression profile of THP-1 derived macrophages cultivated in hyperglycemia and hypoxia. Effect of hyperglycemia and hypoxia on gene expression of inflammatory cytokines The impact of hypoxia and hyperglycemia on the gene expression of TNF- α, IL-1a, IL-6 and GM-CSF was analyzed in detail and compared to the results obtained with the microarray.

Fig 3. Fig 5. Impact of hyperglycemia and hypoxia in activated macrophages on the gene expression of TGF-β, the major wound healing molecule.

Fig 6. Impact of hyperglycemia and hypoxia on the gene expression of SOCS-3 in activated macrophages. Conclusion Hyperglycemia has a negative impact on the wound healing of foot diabetic ulcers.

Supporting information. S1 Table. List of primer used for the RT-PCR. s PDF. Acknowledgments The authors would like to thank Dr Oliver Carroll for his technical guidance in the project, Dana Toncu for editorial and critical assessment of the manuscript, and Mr Anthony Sloan for his editorial assistance in finalizing the manuscript.

References 1. Adeghate J, Nurulain S, Tekes K, Feher E, Kalasz H, Adeghate E. Novel biological therapies for the treatment of diabetic foot ulcers.

Expert Opin Biol Ther. Krzyszczyk P, Schloss R, Palmer A, Berthiaume F. The Role of Macrophages in Acute and Chronic Wound Healing and Interventions to Promote Pro-wound Healing Phenotypes.

Front Physiol. Baltzis D, Eleftheriadou I, Veves A. Pathogenesis and treatment of impaired wound healing in diabetes mellitus: new insights. Adv Ther. Clayton Warren ET. A review of the Pathophysiology, classification, and treatment of foot ulcers in Diabetic Patients.

Clinical Diabetes. View Article Google Scholar 5. Markakis K, Bowling FL, Boulton AJ. The diabetic foot in an overview. Diabetes Metab Res Rev. View Article Google Scholar 6. Armstrong DG, Lavery LA. Diabetic foot ulcers: prevention, diagnosis and classification.

Am Fam Physician. Zhao R, Liang H, Clarke E, Jackson C, Xue M.