당뇨병 환자의 대식세포의 면역기능이 망가지는 기전

[현준]

Sustained high glucose exposure sensitizes macrophage responses to cytokine stimuli but reduces their phagocytic activity 지속적인 높은 포도당 환경의 노출은 대식세포가 사이토카인 자극에 대해서 더 민감하게 반응하도록 만들지만, 반대로 오히려 대식세포의 면역능력은 감소시킨다.

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. 2018 Jul 11;19(1):24. doi: 10.1186/s12865-018-0261-0. PMID: 29996768; PMCID: PMC6042333.

요약

당뇨병 환자의 대식세포는 정상인의 대식세포에 비해 염증반응을 더 많이, 더 민감하게 발생시키지만

항원이나 손상된 세포에 대한 식세포 기능과

손상된 조직을 회복할 수 있는 M2 대식세포 발현이 저하된다.

그러므로 염증만 많이 생기고 염증 이후 회복반응이나 항원제거기능이 잘 일어나지 않게 되어

각종 당뇨병성 합병증이 호발된다.

Abstract

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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.

대식세포는 우리몸의 방어와 항상성을 유지하는 중요한 면역세포이다.

당뇨병에 걸리면 대식세포 및 다른 선천성 면역세포들은 호염증성(pro-inflammatory) 성향을 갖게 된다. 이러한 염증유발 성향때문에 다양한 당뇨병 합병증 발병에 기여하게 된다.

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.

모순적인 부분은 염증반응은 외부로 부터의 침입을 방어하고 회복하는 기전인데, 당뇨병 환자들은 염증이 잘 생기는데 오히려 외부 박테리아 감염이라던지 손상된 조직의 복구 능력이 떨어지게 된다. (염증만 잘 생기고 면역능력, 회복능력은 감소되어 안좋은 점만 부각)

BMDMs:

Bone marrow derived macrophages

HighGlu:

High glucose (30.5 mM)

NormGlu:

Normal glucose (5.5 mM)

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.

골수세포가 고농도의 포도당 환경에 짧게 혹은 길게 노출되었든, 정상 포도당 농도에서 배양되었든 상관없이 골수에서 만들어진 대식세포의 숫자와 표현형에는 큰 차이가 없었다. 즉 고농도의 포도당 환경은 대식세포의 생성 숫자와 phenotype 에 영향을 주지 않음

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.

고농도 포도당에 장기간 노출된 상태에서 생성된 대식세포는 IL-1β(인터류킨), TNFα(종양괴사인자) 의 생성은 증가되었지만 IL-12p40, nitric oxide(NO) 의 생성은 감소되었다. => 염증유발 물질은 많이 만들어지는데, 면역기능을 하는 물질은 적게 만들어짐

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.

고농도 포도당 장기간 노출은 대식세포의 해당 능력을 감소시켰음. 이때 미토콘드리아의 ATP 생산과 산화반응에는 아무런 영향은 없었음.

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.

결론

장기간 고농도 포도당에 노출된 환경에서 골수세포로부터 생성된 대식페노는 사이토카인 자극에 민감하게 반응하여 염증반응을 일으키지만 반면에 식세포작용과 NO생산능력이 감소된다. 이러한 감소는 대식세포의 해당과정 능력의 손상과 연관되어있는 것 같다.

Background

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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 [1].

조직손상과 감염이 있는 부위에서 염증반응이 생기면서 순환중인 단핵구들이 이곳으로 모여서 대식세포로 분화된다. 대식세포의 기능은 외부물질이나 항원을 제거하고 손상된 조직복구를 촉진하는 것이다.

당뇨병은 대식세포와 선천성 면역세포들이 호염증성 형질을 띄게 하는데 이러한 염증성향은 당뇨병의 다양한 합병증의 발병에 기여하는 것으로 보인다.

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 [6]. 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.

당뇨병환자들은 외부 감염으로부터 굉장히 취약하고 대부분 손상회복 능력이 저하되어있다.

Recent evidence suggests that macrophage functions are governed by metabolic reprograming [7]. Metabolic pathways not only provide energy in the form of adenosine triphosphate (ATP), but also regulate macrophage functions [7]. For example, during acute inflammation, the classically activated macrophages (M1) predominately utilize glycolysis, but not mitochondrial oxidative phosphorylation, to support their inflammatory function [8].

급성 염증반응동안 대식세포(M1)은 염증과 관련된 기능을 수행하기 위해 필요한 대부분의 에너지를 해당과정으로부터 얻는다. 미토콘드리아의 산화적 인산화로부터는 잘 얻지 않는다.

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) [7]. 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) [8], whereas succinate enhances the stabilization of HIF1α and supports IL-1β expression‎ [9]. 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.

Results

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The effect of long-term high glucose on bone marrow cell proliferation and differentiation

In diabetes, myeloid progenitor cells are subjected to sustained high glucose conditions. To investigate the impact of the sustained hyperglycaemic environment on macrophage progenitors, we generated BMDMs under high glucose DMEM (HighGlu) conditions. Flow cytometric analysis showed that over 96% of cells were F4/80+ (Fig. 1a). The percentage and absolute number of F4/80+ macrophages were similar in BMDMs generated under normal glucose DMEM ( NormGlu) and HighGlu conditions (Fig. 1a). MTT assay further confirmed that the cell numbers from different groups were also similar (Fig. 1b).

Fig. 1

Ki67 is a nuclear protein necessary for cell proliferation [10]. 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.

당뇨병에서 골수세포는 지속적으로 고농도 포도당 환경에 있다. 이러한 환경이 대식세포의 생성과 분화에 영향을 주는지 확인하기 위해 여러 환경에서 배양해서 테스트해본 결과

포도당 농도와 상관없이 생성된 대식세포의 형질(F4/80+)과 숫자에는 차이가 없었다.

Short-term and long-term high glucose treatment on BMDM immune gene expression‎

Next, we investigated the impact of HighGlu on macrophage gene expression‎. The exposure of naïve BMDMs generated under NormGlu to HighGlu for 24 h (short-term HighGlu) increased IL-1β expression‎ but did not affect the expression‎ of inducible nitric oxide synthase 2 (iNOS), tumour necrosis factor-α (TNFα), interleukin 6 (IL-6), interleukin 12p40 (IL-12p40), arginase-1 (Arg-1), and interleukin 10 (IL-10) (Fig. 2a). When M1 macrophages were exposed to HighGlu for 24 h, the expression‎ of IL-1β was significantly increased (Fig. ​(Fig.2b).2b). Short-term HighGlu treatment did not affect other genes expression‎ in both M1 (Fig. ​(Fig.2b)2b) and M2 macrophages (Fig. ​(Fig.2c).2c). NO production was also not affected (Fig. ​(Fig.2d).2d). The results suggest that short-term HighGlu enhances macrophage pro-inflammatory response.

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

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. ​(Fig.2e).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. ​(Fig.2f).2f). Long-term HighGlu treatment also reduced the expression‎ of iNOS, although not significantly (Fig. ​(Fig.2f).2f). In contrast with the iNOS expression‎, NO production was significantly lower in long-term HighGlu treated M1 macrophages (Fig. ​(Fig.2h).2h). Interestingly, IL4-induced upregulation of Arg-1 and IL-10 was significantly enhanced in long-term HighGlu treated BMDMs (Fig. ​(Fig.2g).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.

To investigate if the increased IL-1β mRNA expression‎ in HighGlu-treated macrophages resulted in higher levels of protein secretion, IL-1β levels in culture supernatants were measured using ELISA. The results show that the levels of IL-1β are undetectable in naïve BMDMs (data not shown). LPS + IFNγ stimulation and high glucose significantly increased IL-1β production (Fig. 3a-​-bb).

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

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. ​(Fig.3c),3c), whereas long-term HighGlu significantly increased the expression‎ of TLR-4, interferon gamma receptor 1 (IFNγR1) and interferon gamma receptor 2 (IFNγR2) (Fig. ​(Fig.3d).3d). Interestingly, the expression‎ levels of interleukin 4 receptor, alpha (IL4ra) remained unchanged in both short-term and long-term HighGlu treated macrophages (Fig. ​(Fig.3c3c-​-dd).

The effect of long-term high glucose on BMDM phagocytosis and bactericidal function

The evident difference between short-term and long-term HighGlu on macrophage response to cytokine stimulation prompted us to further investigate other macrophage functions such as phagocytosis. Using the pHrodo S. aureus bioparticles phagocytosis assay, we did not observe any difference between macrophages treated with or without short-term HighGlu (Additional file 1: Figure S1). However, in macrophages generated under HighGlu significantly reduced phagocytosis was observed after 1.5 h and 2 h of bioparticle incubation compared with BMDMs in NormGlu and Mannitol conditions (Fig. 4a). Long-term Mannitol treatment did not affect macrophage phagocytosis (Fig. 4a).

고농도 포도당 환경에 노출되었던 대식세포는 식세포 작용이 감소,저하되지 않았음

그러나 고농도 포도당 환경의 골수세포에서 생성된 대식세포는 식세포 작용이 감소되었음.

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

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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.

당뇨병 쥐에서 추출한 대식세포의 식세포 능력이 감소되어있었다.

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.

장기간의 고농도 포도당 환경에서 생성된 대식세포의 식균능력은 저하된다. 따라서 박테리아가 다른 대식세포 그룹에 비해 더 많이 살아남아 있었다.

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).

고농도 포도당 환경에서 생성된 대식세포는 어떤 부분에서 차이가 생기는 건지 조사했을때, 대식세포의 표면에 있는 수용체중에 CD36 및 CD206의 발현이 정상 대식세포보다 유의하게 낮았다. -> 고농도 포도당이 대식세포 생성에 영향을 주어 비정상? 대식세포가 만들어지게 한다.

The effect of long-term high glucose on macrophage glucose metabolism and mitochondrial oxygen consumption

Immune cells’ function is governed by their metabolisms [11]. We hypothesize that long-term HighGlu treatment may affect macrophage function through regulating their metabolic pathways. In the Glycolysis Stress assay, BMDMs were incubated in basal medium (medium without added glucose, supplemented with glutamine) for 1 h followed by the various stimulations depicted in Fig. 5a. The non-glycolytic (baseline) extracellular acidification rate (ECAR) of naïve BMDMs in all culture conditions was similar (Fig. 5a). The addition of glucose and subsequently oligomycin resulted in a rapid increase in ECAR (Fig. 5a). Both short-term (24 h, Fig. 5a, b) and long-term (7 days, Fig. 5c, d) HighGlu treatment significantly reduced BMDM response to glucose and oligomycin stimulations. Specifically, the short-term HighGlu treatment reduced glycolysis and glycolytic capacity (Fig. 5b); whereas long-term HighGlu reduced glycolytic capacity and glycolytic reserve (Fig. 5d).

고농도 포도당에 노출된 환경에서 생성된 대식세포의 해당과정 능력이 저하되어 있다. 단기 고농도 포도당 환경에서 생성된 대식세포는 해당과정 및 해당과정 용량이 저하되었고 장기간 노출되었을 경우 해당과정용량 뿐만아니라 해당과정 예비율도 떨어져있었다.

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

Discussion

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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.

고농도 포도당 환경에서 생성된 대식세포는 정상 포도당 환경에서 생성된 대식세포와 여러가지 차이가 생긴다.

정상 대식세포와 HighGlu 대식세포 둘다 사이토카인에 대해서 유사하게 염증성으로 반응한다. 하지만 HighGLu 대식세포의 경우 식세포능력과 손상된 조직 회복능력은 감소되어 있다.

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 [13]. The populations of circulating F4/80+, CD11b+, CCR2+ or Ly6G+ cells in diabetic mice were reported to be reduced in some studies [14], but increased in others [6]. 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 [17], 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).

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 [29]. TNFα and IL-1β were also higher in cultures of peritoneal macrophages in STZ-induced diabetic rats comparing to control rats [23]. 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 [32] 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.

당뇨병 쥐에서 채취한 대식세포가 대조군에 비해 더 많은 TNF알파와 IL-1베타를 생성하였다. 즉 호염증성이다.

장기간의 고농도 포도당 노출은 대식세포가 사이토카인 자극에 대해서 더 민감하게 반응하도록 만든다.

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 [33], 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.

고농도 포도당 환경에서 골수세포로 부터 생성된 대식세포의 미토콘드리아 기능은 손상되지 않지만 해당과정 능력과 해당 예비력이 저하된다.

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 [34]. 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 [35].

Conclusions

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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.

이 연구에서 장시간 고농도 포도당 환경에서 생성된 대식세포는 사이토카인 자극에 대해서 민감하게 반응한다. 하지만 대식세포의 식세포 능력과 박테리아에 대한 살균 기능이 저하되었다.

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정리

당뇨병 환자는 염증에 의한 각종 합병증에 시달린다. 당뇨병 환자의 대식세포의 특징은 염증 유발 요인에 대해 더욱 민감하게 반응하여 염증이 더 잘 일어나게 된다. 근데 모순적이게도 염증반응은 잘 일어나지만 정작 염증으로 없애야 할 외부항원이나, 염증이후 손상된 조직 회복 기능은 오히려 저하되어 있다.

그에 대한 원인을 실험한 논문으로, 당뇨병 환자의 몸상태는 혈당조절이 잘 안되므로 혈당이 높아져있는, 고농도 포도당 상태이다. 이러한 고농도 포도당 상태에서 골수세포로 부터 생성된 대식세포는 정상환경 대식세포와 차이가 생긴다. 그러한 차이점은 아래와 같다.

1) 사이토카인에 더 민감하게 반응하여 TNF알파 IL-1베타 등 호염증성 사이토카인을 더 많이 분비하게 된다. 즉 호염증성 성향이 더 강해진다.

2) 식세포작용(Phagocytosis)과 산화질소(NO) 생성능력이 저하된다. 이는 대식세포가 원활한 염증반응과 면역반응을 위해서는 해당과정(Glycolysis)을 통해서 에너지를 얻어야 하는데 고농도 포도당 환경에서 생성된 대식세포의 해당과정 능력이 손상되고 저하되어 있기 때문이다.

3) 대식세포에는 염증반응을 촉진하는 M1 대식세포와 회복을 촉진하는 M2 대식세포가 있음. 고농도 포도당 환경 즉 당뇨병 환경에서는 염증반응시 M1 대식세포가 많아지고 M2 대식세포의 활동과 생성이 저하되어 회복능력이 손상됨 (타 논문 내용 참고함 https://pubmed.ncbi.nlm.nih.gov/24057002/ Diabetes induces stable intrinsic changes to myeloid cells that contribute to chronic inflammation during wound healing in mice )

위와 같은 이유로 고농도 포도당 환경인 당뇨병 환자는 염증은 잘 일어나지만 항원을 없애는 능력과 손상된 조직을 회복하는 능력이 저하되어 다양한 당뇨병성 합병증이 호발하게 된다.