Emerging evidence suggests that metabolic adaptation is a vital hallmark and prerequisite for macrophage phenotype transition. Pyruvate kinase M2 (PKM2) is an essential molecular determinant of metabolic adaptions in pro-inflammatory macrophages. Post-translational modifications play a central role in the regulation of PKM2. However, doubt remains on whether lactylation in PKM2 exists and how lactylation modulates the function of PKM2. For the first time, our study reports that lactate inhibits the Warburg effect by activating PKM2, promoting the transition of pro-inflammatory macrophages towards a reparative phenotype. We identify PKM2 as a lactylation substrate and confirm that lactylation occurs mainly at the K62 site. We find that lactate increases the lactylation level of PKM2, which inhibits its tetramer-to-dimer transition, promoting its pyruvate kinase activity and reducing nuclear distribution. In short, our study reports a novel post-translational modification type in PKM2 and clarifies its potential role in regulating inflammatory metabolic adaptation in pro-inflammatory macrophages.

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.

Acute lung injury (ALI) is the leading cause of death in sepsis patients. Exosomes participate in the occurrence and development of ALI by regulating endothelial cell inflammatory response, oxidative stress and apoptosis, causing serious pulmonary vascular leakage and interstitial edema. The current study investigated the effect of exosomal miRNAs on endothelial cells during sepsis. We found a significant increase in miR-1-3p expression in cecal ligation and puncture (CLP) rats exosomes sequencing and sepsis patients’ exosomes, and lipopolysaccharide (LPS)-stimulated human umbilical vein endothelial cells (HUVECs) in vitro. However, the specific biological function of miR-1-3p in ALI remains unknown. Therefore, mimics or inhibitors of miR-1-3p were transfected to modulate its expression in HUVECs. Cell proliferation, apoptosis, contraction, permeability, and membrane injury were examined via cell counting kit-8 (CCK-8), flow cytometry, phalloidin staining, Transwell assay, lactate dehydrogenase (LDH) activity, and Western blotting. The miR-1-3p target gene was predicted with miRNA-related databases and validated by luciferase reporter. Target gene expression was blocked by siRNA to explore the underlying mechanisms. The results illustrated increased miR-1-3p and decreased stress-associated endoplasmic reticulum protein 1 (SERP1) expression both in vivo and in vitro. SERP1 was a direct target gene of miR-1-3p. Up-regulated miR-1-3p inhibits cell proliferation, promotes apoptosis and cytoskeleton contraction, increases monolayer endothelial cell permeability and membrane injury by targeting SERP1, which leads to dysfunction of endothelial cells and weakens vascular barrier function involved in the development of ALI. MiR-1-3p and SERP1 may be promising therapeutic candidates for sepsis-induced lung injury.

Dexmedetomidine (Dex) has been shown to elicit cardio-protective effects in sepsis. The aim of this study was to investigate the role of autophagy in the protective effects of Dex and its possible mechanism in vivo and vitro. 6-8-week-old male Wistar rats were performed cecal ligation puncture (CLP) and administered 0.9% saline (CLP group), 50 μg/kg Dex (Dex group), Dex plus chloroquine (20 mg/kg; Dex + CQ group), or 40 μg/kg methyllycaconitin (Dex + MLA group), or 25 μM LY294002 (Dex + LY294002 group). After study, cardiac histology, cardiac function, level of autophagy, cardiomyocytes apoptosis and inflammatory mediators including protein IL-1β, IL-6, and TNF-α were measured. The LPS induced-H9C2 cardiomyocytes were treated with Dex, Dex + CQ and detected for cell apoptosis, autophagy level and cell cycle. CLP-induced sepsis resulted in cardiac dysfunction, apoptosis, and inflammatory response. Dex exhibited protective effects on the myocardium by the induction of myocardial autophagy and ameliorated the LPS-induced blockade of autophagic flux in H9C2 cells. CQ was found to significantly inhibit Dex-mediated protection of myocardial apoptosis and inflammation. CLP rats treated with Dex in combination with MLA, an antagonist of α7 nicotinic acetylcholine receptor (α7nAChR), exhibited decreased autophagy and increased inflammation and cell death, identifying α7nAchR was involved in the Dex-mediated pathway. In addition, we found that the PI3K/Akt pathway is involved in Dex-mediated autophagy and convergent with α7nAChR-mediated stimulation of autophagy response. For the first time, these data indicate that autophagy is central in Dex-mediated cardio-protection in sepsis. These observations provide the foundation for further study, and may serve as the basis for innovative therapeutic strategies against septic myocardial dysfunction.

Besides regulating glucose levels, insulin has been reported to participate actively in many other functions such as modulating inflammatory reaction. In this study we investigated how insulin application topically would effort the diabetic wound healing process. We found that the excessive expression of insulin degrading enzyme in diabetic skin led to the insufficient insulin level in diabetic skin during the wound healing, which ultimately reduced the recovery rate of diabetic wounds. We confirmed the topical insulin application could reverse the impaired inflammation reaction in diabetic wound environment and promote diabetic wounds healing. Our study revealed that insulin promoted neutrophils apoptosis and following triggered macrophages phenotype polarization. Both in vivo and in vitro studies verified that insulin reestablished the damaged neutrophils phagocytosis function and promoted the process of phagocytosis induced apoptosis of neutrophils. Furthermore, we found that the insulin treatment also promoted macrophages efferocytosis of the apoptosed neutrophils and thus induced macrophages polarized from M1 to M2. In conclusion, our studies proved that the exogenous application of insulin could improve the diabetic wound healing via the normalization of inflammatory response.