Targeting the NLRP3 inflammasome to improve healing of diabetic wounds

Introduction. To investigate the contribution and mechanism of NLRP3 inflammasome expression in human wounds in diabetes mellitus and in high glucose induced macrophages. Methods. In the present study, we compared the expression of NLRP3 inflammasome in debridement wound tissue from diabetic and nondiabetic patients. We also examined whether high glucose induces NLRP3 inflammasome expression in cultures THP-1-derived macrophages and the influence on IL-1β expression. Results. The expressions of NLRP3, caspase1, and IL-1β, at both the mRNA and protein level, were significantly higher in wounds of diabetic patients compared with nondiabetic wounds (P<0.05). High glucose induced a significant increase in NLRP3 inflammasome and IL-1β expression in THP-1-derived macrophages. M1 macrophage surface marker with CCR7 was significantly upregulated after high glucose stimulation. SiRNA-mediated silencing of NLRP3 expression downregulates the expression of IL-1β. Conclusion. The higher expression of NLRP3, caspase1, and secretion of IL-1β, signaling, and activation might contribute to the hyperinflammation in the human diabetic wound and in high glucose induced macrophages. It may be a novel target to treat the DM patients with chronic wound.

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651 NLRP3 Activation Induced by Neutrophil Extracellular Traps Sustains Inflammatory Response in the Diabetic Wound

Journal of Burn Care & Research ◽

10.1093/jbcr/iraa024.270 ◽

2020 ◽

Vol 41 (Supplement_1) ◽

pp. S171-S172

Author(s):

Dan Liu

Keyword(s):

Wound Healing ◽

Inflammatory Response ◽

Nlrp3 Inflammasome ◽

Healing Process ◽

Dnase I ◽

Cell Infiltration ◽

Type I ◽

Diabetic Wound ◽

Extracellular Traps ◽

Diabetic Wounds

Abstract Introduction Persistent inﬂammatory response in the diabetic wound impairs the healing process, resulting in significant morbidity and mortality. Mounting evidence indicates that the activation of NLRP3 inflammasome in macrophages (MF) contributes to the sustained inﬂammatory response and impaired wound healing associated with diabetes. However, the main trigger of NLRP3 inflammasome in the wounds is not known. Neutrophils, as sentinels of the innate immune system and key stimulators of MF, are immune cells that play the main role in the early phase of healing. Neutrophils release extracellular traps (NETs) as defense against pathogens. On the other hand, NETs induce tissue damage. NETs have been detected in the diabetic wound and implicated in the impaired healing process, but the mechanism of NETs suspend wound healing are elusive. Methods WB, immunofluorescence and immunocoprecipitation were used to detect the expression of NETs, NLRP3 inflammasome and IL-1b in diabetic foot ulcer wounds and the expression and activation of NLRP3 inflammsome after lowering the level of NETs. The role of NETs in activating macrophage inflammsome was verified and its mechanism was explored. Induced type I diabetic rats by STZ injection to detect the expression of NETs in the wound and observe the effects of exogenous DNase I on the expression of inflammasome and IL-1b in the wound, inflammatory cell infiltration and wound healing. Results NLRP3 and NETs are elevated in human and rat diabetic wounds. NETs overproduced in the diabetic wounds triggered NLRP3 inflammasome activation and IL-1b release. NETs up-regulated NLRP3 and pro-IL-1b levels via the TLR-4/TLR-9/NF-kB signalling pathway. NETs elicited ROS, which facilitated the association between NLRP3 and TXNIP, and activated the NLRP3 inflammasome. DNase I alleviated the activation of NLRP3 inflammasome, regulated the immune cell infiltration, and accelerated wound healing. Conclusions NETs contribute to the activation of NLRP3 inflammasome and sustained inflammatory response in the diabetic wound. NET digestion by DNase I alleviated the activation of NLRP3 inflammasome and accelerated wound healing. Applicability of Research to Practice

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Autophagy Inhibition Contributes to ROS-Producing NLRP3-Dependent Inflammasome Activation and Cytokine Secretion in High Glucose-Induced Macrophages

Cellular Physiology and Biochemistry ◽

10.1159/000480367 ◽

2017 ◽

Vol 43 (1) ◽

pp. 247-256 ◽

Cited By ~ 31

Author(s):

Jiezhi Dai ◽

Xiaotian Zhang ◽

Li Li ◽

Hua Chen ◽

Yimin Chai

Keyword(s):

Nlrp3 Inflammasome ◽

High Glucose ◽

Cytokine Secretion ◽

Inflammasome Activation ◽

Ros Production ◽

Oxygen Species ◽

Nlrp3 Inflammasome Activation ◽

Diabetic Wounds ◽

Autophagy Inhibition ◽

Inflammasome Activity

Background: Type 2 diabetes is a persistent inflammatory response that impairs the healing process. We hypothesized that stimulation with high glucose following a pro-inflammatory signal would lead to autophagy inhibition, reactive oxygen species (ROS) production and eventually to the activation of the Nod-like receptor protein (NLRP) -3. Methods: Macrophages were isolated from human diabetic wound. We measured the expression of NLRP3, caspase1 and interleukin-1 beta (IL-1β) by western blot and real-time PCR, and the surface markers on cells by flow cytometry. THP-1-derived macrophages exposed to high glucose were applied to study the link between autophagy, ROS and NLRP3 activation. LC3-II, P62, NLRP3 inflammation and IL-1β expression were measured by western blot and real-time PCR. ROS production was measured with a Cellular Reactive Oxygen Species Detection Assay Kit. Results: Macrophages isolated from diabetic wounds exhibited a pro-inflammatory phenotype, including sustained NLRP3 inflammasome activity associated with IL-1β secretion. Our data showed that high glucose inhibited autophagy, induced ROS production, and activated NLRP3 inflammasome and cytokine secretion in THP-1-derived macrophages. To study high glucose-induced NLRP3 inflammasome signalling, we performed studies using an autophagy inducer, a ROS inhibitor and a NLRP3 inhibitor and found that all reduced the NLRP3 inflammasome activation and cytokine secretion. Conclusion: Sustained NLRP3 inflammasome activity in wound-derived macrophages contributes to the hyper-inflammation in human diabetic wounds. Autophagy inhibition and ROS generation play an essential role in high glucose-induced NLRP3 inflammasome activation and cytokine secretion in macrophages.

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NLRP3 activation induced by neutrophil extracellular traps sustains inflammatory response in the diabetic wound

Clinical Science ◽

10.1042/cs20180600 ◽

2019 ◽

Vol 133 (4) ◽

pp. 565-582 ◽

Cited By ~ 15

Author(s):

Dan Liu ◽

Peilang Yang ◽

Min Gao ◽

Tianyi Yu ◽

Yan Shi ◽

...

Keyword(s):

Wound Healing ◽

Inflammatory Response ◽

Nlrp3 Inflammasome ◽

Healing Process ◽

Receptor Protein ◽

Main Role ◽

Diabetic Wound ◽

Impaired Wound Healing ◽

Extracellular Traps ◽

Diabetic Wounds

Abstract Persistent inflammatory response in the diabetic wound impairs the healing process, resulting in significant morbidity and mortality. Mounting evidence indicate that the activation of Nod-like receptor protein (NLRP) 3 inflammasome in macrophages (MΦ) contributes to the sustained inflammatory response and impaired wound healing associated with diabetes. However, the main trigger of NLRP3 inflammasome in the wounds is not known. Neutrophils, as sentinels of the innate immune system and key stimulators of MΦ, are immune cells that play the main role in the early phase of healing. Neutrophils release extracellular traps (NETs) as defense against pathogens. On the other hand, NETs induce tissue damage. NETs have been detected in the diabetic wound and implicated in the impaired healing process, but the mechanism of NETs suspend wound healing and its role in fostering inflammatory dysregulation are elusive. Here, we report that NLRP3 and NETs production are elevated in human and rat diabetic wounds. NETs overproduced in the diabetic wounds triggered NLRP3 inflammasome activation and IL-1β release in MΦ. Furthermore, NETs up-regulated NLRP3 and pro-IL-1β levels via the TLR-4/TLR-9/NF-κB signaling pathway. They also elicited the generation of reactive oxygen species, which facilitated the association between NLRP3 and thioredoxin-interacting protein, and activated the NLRP3 inflammasome. In addition, NET digestion by DNase I alleviated the activation of NLRP3 inflammasome, regulated the immune cell infiltration, and accelerated wound healing in diabetic rat model. These findings illustrate a new mechanism by which NETs contribute to the activation of NLRP3 inflammasome and sustained inflammatory response in the diabetic wound.

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Inflammageing in the cardiovascular system: mechanisms, emerging targets, and novel therapeutic strategies

Clinical Science ◽

10.1042/cs20191213 ◽

2020 ◽

Vol 134 (17) ◽

pp. 2243-2262

Author(s):

Danlin Liu ◽

Gavin Richardson ◽

Fehmi M. Benli ◽

Catherine Park ◽

João V. de Souza ◽

...

Keyword(s):

Cardiovascular Diseases ◽

Cardiovascular System ◽

Nlrp3 Inflammasome ◽

Molecular Mechanisms ◽

Molecular Targets ◽

Therapeutic Interventions ◽

Low Grade ◽

Cardiovascular Research ◽

Inflammatory Processes ◽

Ach Receptors

Abstract In the elderly population, pathological inflammation has been associated with ageing-associated diseases. The term ‘inflammageing’, which was used for the first time by Franceschi and co-workers in 2000, is associated with the chronic, low-grade, subclinical inflammatory processes coupled to biological ageing. The source of these inflammatory processes is debated. The senescence-associated secretory phenotype (SASP) has been proposed as the main origin of inflammageing. The SASP is characterised by the release of inflammatory cytokines, elevated activation of the NLRP3 inflammasome, altered regulation of acetylcholine (ACh) nicotinic receptors, and abnormal NAD+ metabolism. Therefore, SASP may be ‘druggable’ by small molecule therapeutics targeting those emerging molecular targets. It has been shown that inflammageing is a hallmark of various cardiovascular diseases, including atherosclerosis, hypertension, and adverse cardiac remodelling. Therefore, the pathomechanism involving SASP activation via the NLRP3 inflammasome; modulation of NLRP3 via α7 nicotinic ACh receptors; and modulation by senolytics targeting other proteins have gained a lot of interest within cardiovascular research and drug development communities. In this review, which offers a unique view from both clinical and preclinical target-based drug discovery perspectives, we have focused on cardiovascular inflammageing and its molecular mechanisms. We have outlined the mechanistic links between inflammageing, SASP, interleukin (IL)-1β, NLRP3 inflammasome, nicotinic ACh receptors, and molecular targets of senolytic drugs in the context of cardiovascular diseases. We have addressed the ‘druggability’ of NLRP3 and nicotinic α7 receptors by small molecules, as these proteins represent novel and exciting targets for therapeutic interventions targeting inflammageing in the cardiovascular system and beyond.

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