Peroxiredoxin 3 has a crucial role in the macrophage polarization by regulating mitochondrial homeostasis.

Acute lung injury (ALI) is one of the life-threatening complications of sepsis, and macrophage polarization plays a crucial role in the sepsis-associated ALI. However, the regulatory mechanisms of macrophage polarization in ALI and in the development of inflammation are largely unknown. In this study, we demonstrated that macrophage polarization occurs in sepsis-associated ALI and is accompanied by mitochondrial dysfunction and inflammation, and a decrease of PRDX3 promotes the initiation of macrophage polarization and mitochondrial dysfunction. Mechanistically, PRDX3 overexpression promotes M1 macrophages to differentiate into M2 macrophages, and enhances mitochondrial functional recovery after injury by reducing the level of glycolysis and increasing TCA cycle activity. In conclusion, we identified PRDX3 as a critical hub integrating oxidative stress, inflammation, and metabolic reprogramming in macrophage polarization. The findings illustrate an adaptive mechanism underlying the link between macrophage polarization and sepsis-associated ALI.

Ferritin-mediated neutrophil extracellular traps formation and cytokine storm via macrophage scavenger receptor in sepsis-associated lung injury.

BACKGROUND: Sepsis is a severe systemic inflammatory disorder manifested by a dysregulated immune response to infection and multi-organ failure. Numerous studies have shown that elevated ferritin levels exist as an essential feature during sepsis and are able to suggest patients' prognoses. At the same time, the specific mechanism of ferritin-induced inflammatory injury remains unclear. METHODS: Hyper-ferritin state during inflammation was performed by injecting ferritin into a mouse model and demonstrated that injection of ferritin could induce a systemic inflammatory response and increase neutrophil extracellular trap (NET) formation.Padi4-/-, Elane-/- and Cybb-/- mice were used for the NETs formation experiment. Western blot, immunofluorescence, ELISA, and flow cytometry examined the changes in NETs, inflammation, and related signaling pathways. RESULTS: Ferritin induces NET formation in a peptidylarginine deiminase 4 (PAD4), neutrophil elastase (NE), and reactive oxygen species (ROS)-dependent manner, thereby exacerbating the inflammatory response. Mechanistically, ferritin induces the expression of neutrophil macrophage scavenger receptor (MSR), which promotes the formation of NETs. Clinically, high levels of ferritin in patients with severe sepsis correlate with NETs-mediated cytokines storm and are proportional to the severity of sepsis-induced lung injury. CONCLUSIONS: In conclusion, we demonstrated that hyper-ferritin can induce systemic inflammation and increase NET formation in an MSR-dependent manner. This process relies on PAD4, NE, and ROS, further aggravating acute lung injury. In the clinic, high serum ferritin levels are associated with elevated NETs and worse lung injury, which suggests a poor prognosis for patients with sepsis. Our study indicated that targeting NETs or MSR could be a potential treatment to alleviate lung damage and systemic inflammation during sepsis. Video Abstract.

TFAM-Mediated mitochondrial transfer of MSCs improved the permeability barrier in sepsis-associated acute lung injury.

Vascular endothelial cell barrier disruption is a hallmark of sepsis-induced acute lung injury (ALI). Mesenchymal stem cells (MSCs)-based therapy has been regarded as a promising treatment for repairing injured lungs, and mitochondrial transfer was shown to be important for the therapeutic effects of MSCs. Here we investigated the ability of MSCs to modulate endothelial barrier integrity through mitochondrial transfer in sepsis-induced ALI. We found that mitochondrial transfer from MSCs to LPS-induced PMVECs through forming tunneling nanotubes (TNTs). Due to the inhibition of TNTs (using LAT-A), MSCs-mediated reparation on PMVECs functions, including cell apoptosis, MMP, ATP generation, TEER level and monolayer permeability of FITC-dextran were greatly inhibited. In addition, silencing of mitochondrial transcription factor A (TFAM) in MSCs could also partly inhibit the TNTs formation and aggravate the LPS-induced mitochondrial dysfunction and permeability barrier in PMVECs. Furthermore, the LPS-induced pulmonary edema and higher pulmonary vascular permeability were alleviated by MSCs while that of lung tissue bounced back after MSCs were pre-incubated by LAT-A and or down-regulation of TFAM. Therefore, we firstly revealed that regulation of TFAM expression in MSCs played a critical role to improve the permeability barrier of PMVECs by TNTs mediating mitochondrial transfer in sepsis-associated ALI. This study provided a new therapeutic strategy for the treatment of sepsis-induced ALI.

Histone lactylation-regulated METTL3 promotes ferroptosis via m6A-modification on ACSL4 in sepsis-associated lung injury.

Elevated lactate levels are a significant biomarker of sepsis and are positively associated with sepsis-related mortality. Sepsis-associated lung injury (ALI) is a leading cause of poor prognosis in clinical patients. However, the underlying mechanisms of lactate's involvement in sepsis-associated ALI remain unclear. In this study, we demonstrate that lactate regulates N6-methyladenosine (m6A) modification levels by facilitating p300-mediated H3K18la binding to the METTL3 promoter site. The METTL3-mediated m6A modification is enriched in ACSL4, and its mRNA stability is regulated through a YTHDC1-dependent pathway. Furthermore, short-term lactate stimulation upregulates ACSL4, which promotes mitochondria-associated ferroptosis. Inhibition of METTL3 through knockdown or targeted inhibition effectively suppresses septic hyper-lactate-induced ferroptosis in alveolar epithelial cells and mitigates lung injury in septic mice. Our findings suggest that lactate induces ferroptosis via the GPR81/H3K18la/METTL3/ACSL4 axis in alveolar epithelial cells during sepsis-associated ALI. These results reveal a histone lactylation-driven mechanism inducing ferroptosis through METTL3-mediated m6A modification. Targeting METTL3 represents a promising therapeutic strategy for patients with sepsis-associated ALI.

A purified membrane protein from Akkermansia muciniphila blunted the sepsis-induced acute lung injury by modulation of gut microbiota in rats.

The gut microbiota has been implicated in the pathogenesis and progression of sepsis. Akkermansia muciniphila is considered to be a promising probiotic with reduced abundance in cecal ligation and puncture (CLP)-induced sepsis model, and its specific outer membrane protein (Amuc_1100) can partially recapitulate the probiotic function of Akkermansia muciniphila. However, its role in sepsis is unclear. This study aimed to investigate the effect of Amuc_1100 on the gut microbiota of septic rats, thereby improving the prognosis of septic acute lung injury (ALI). A total of 42 adult Sprague-Dawley (SD) rats were randomly divided into three groups: the sham control (SC group), the septic ALI induced by CLP method (CLP group), and administered Amuc_1100 by oral gavage (3 µg/d) for 7 d before the CLP procedure (AMUC group). The survival of the three groups was recorded and the feces and lung tissues of rats were collected 24 h after treatment for 16S rRNA sequencing and histopathological evaluation. Oral administration of Amuc_1100 improved the survival rate and alleviated lung histopathological damage induced by sepsis. Serum levels of pro-inflammatory cytokines and chemokines were substantially attenuated. Amuc_1100 significantly increased the abundance of some beneficial bacteria in septic rats. Additionally, the Firmicutes/Bacteroidetes ratio was low in septic rats, which was partially corrected by increasing Firmicutes and decreasing Bacteroidetes after oral administration of Amuc_1100 (p < 0.05). In addition, Escherichia-Shigella, Bacteroides, and Parabacteroides were relatively enriched in septic rats, while in the AMUC group, their abundance was restored to levels similar to that of the healthy group. Amuc_1100 protects against sepsis by enhancing beneficial bacteria and reducing potential pathogenic bacteria. These findings indicate that Amuc_1100 can blunt CLP-induced ALI through the modulation of gut microbiota, thereby providing a new promising therapeutic target in sepsis.

Effect of metformin on sepsis-associated acute lung injury and gut microbiota in aged rats with sepsis.

Background: Recent studies reported the association between the changes in gut microbiota and sepsis, but there is unclear for the gut microbes on aged sepsis is associated acute lung injury (SALI), and metformin treatment for the change in gut microbiota. This study aimed to investigate the effect of metformin on gut microbiota and SALI in aged rats with sepsis. It also explored the therapeutic mechanism and the effect of metformin on aged rats with SALI. Methods: Aged 20-21 months SD rats were categorized into three groups: sham-operated rats (AgS group), rats with cecal ligation and puncture (CLP)-induced sepsis (AgCLP group), and rats treated with metformin (100 mg/kg) orally 1 h after CLP treatment (AgMET group). We collected feces from rats and analyzed them by 16S rRNA sequencing. Further, the lung samples were collected for histological analysis and quantitative real-time PCR (qPCR) assay and so on. Results: This study showed that some pathological changes occurring in the lungs of aged rats, such as hemorrhage, edema, and inflammation, improved after metformin treatment; the number of hepatocyte death increased in the AgCLP group, and decreased in the AgMET group. Moreover, metformin relieved SALI inflammation and damage. Importantly, the gut microbiota composition among the three groups in aged SALI rats was different. In particular, the proportion of E. coli and K. pneumoniae was higher in AgCLP group rats than AgS group rats and AgMET group rats; while metformin could increase the proportion of Firmicutes, Lactobacillus, Ruminococcus_1 and Lactobacillus_johnsonii in aged SALI rats. Moreover, Prevotella_9, Klebsiella and Escherichia_Shigella were correlated positively with the inflammatory factor IL-1 in the lung tissues; Firmicutes was correlated negatively with the inflammatory factor IL-1 and IL-6 in the lung tissues. Conclusions: Our findings suggested that metformin could improve SALI and gut microbiota in aged rats, which could provide a potential therapeutic treatment for SALI in aged sepsis.

Cannabinoid receptor 2 alleviates sepsis-associated acute lung injury by modulating maturation of dendritic cells.

BACKGROUND: Dendritic cells (DCs) play a key role in a variety of inflammatory lung diseases, but their role in sepsis-associated acute lung injury (SA-ALI) is currently not been illuminated. Cannabinoid receptor 2 (CNR2) has been reported to regulate the DCs maturation. However, whether the CNR2 in DCs contributes to therapeutic therapy for SA-ALI remain unclear. In current study, the role of CNR2 on DCs maturation and inflammatory during SA-ALI is to explored. METHODS: First, the CNR2 level was analyzed in isolated Peripheral Blood Mononuclear Cells (PBMCs) and Bronchoalveolar Lavage Fluid (BALF) from patient with SA-ALI by qRT-PCR and flow cytometry. Subsequently, HU308, a specific agonist of CNR2, and SR144528, a specific antagonist of CNR2, were introduced to explore the function of CNR2 on DCs maturation and inflammatory during SA-ALI. Finally, CNR2 conditional knockout mice were generated to further confirm the function of DCs maturation and Inflammation during SA-ALI. RESULTS: First, we found that the expression of CNR2 on DCs was decreased in patient with SA-ALI. Besides, the result showed HU308 could decrease the maturation of DCs and the level of inflammatory cytokines, simultaneously reduce pulmonary pathological injury after LPS-induced sepsis in mice. In contrast of HU308, SR144528 exhibits opposite function of DCs maturate, inflammatory cytokines and lung pathological injury. Furthermore, comparing with SR144528 treatment, similar results were obtained in DCs specific CNR2 knockout mice after LPS treatment. CONCLUSION: CNR2 could alleviate SA-ALI by modulating maturation of DCs and inflammatory factors levels. Targeting CNR2 signaling specifically in DCs has therapeutic potential for the treatment of SA-ALI.

3-Hydroxybutyrate ameliorates sepsis-associated acute lung injury by promoting autophagy through the activation of GPR109α in macrophages.

BACKGROUND: Sepsis is a systemic inflammatory disease caused by multiple pathogens, with the most commonly affected organ being the lung. 3-Hydroxybutyrate plays a protective role in inflammatory diseases through autophagy promotion; however, the exact mechanism remains unexplored. METHOD: Our study used the MIMIC-III database to construct a cohort of ICU sepsis patients and figure out the correlation between the level of ketone bodies and clinical prognosis in septic patients. In vivo and in vitro models of sepsis were used to reveal the role and mechanism of 3-hydroxybutyrate in sepsis-associated acute lung injury (sepsis-associated ALI). RESULT: Herein, we observed a strong correlation between the levels of ketone bodies and clinical prognosis in patients with sepsis identified using the MIMIC- III database. In addition, exogenous 3-hydroxybutyrate supplementation improved the survival rate of CLP-induced sepsis in mice by promoting autophagy. Furthermore, 3-hydroxybutyrate treatment protected against sepsis-induced lung damage. We explored the mechanism underlying these effects. The results indicated that 3-hydroxybutyrate upregulates autophagy levels by promoting the transfer of transcription factor EB (TFEB) to the macrophage nucleus in a G-protein-coupled receptor 109 alpha (GPR109α) dependent manner, upregulating the transcriptional level of ultraviolet radiation resistant associated gene (UVRAG) and increasing the formation of autophagic lysosomes. CONCLUSION: 3-Hydroxybutyrate can serve as a beneficial therapy for sepsis-associated ALI through the upregulation of autophagy. These results may provide a basis for the development of promising therapeutic strategies for sepsis-associated ALI.

Neutrophil extracellular traps mediate m6A modification and regulates sepsis-associated acute lung injury by activating ferroptosis in alveolar epithelial cells.

Neutrophil extracellular traps (NETs) production is a major strategy employed by polymorphonuclear neutrophils (PMNs) to fight against microbes. NETs have been implicated in the pathogenesis of various lung injuries, although few studies have explored NETs in sepsis-associated acute lung injury (SI-ALI). Here, we demonstrate a major contribution of NETs to the pathology of sepsis-associated ALI by inducing ferroptosis of alveolar epithelial cells. Using both in vitro and in vivo studies, our findings show enhanced NETs accumulation in sepsis-associated ALI patients and mice, as well as the closely related upregulation of ferroptosis, the induction of which depends on METTL3-induced m6A modification of GPX4. Using a CLP-induced sepsis-associated ALI mouse model established with METTL3-/- versus WT mice, in addition to METTL3 knockout and overexpression in vitro, we elucidated and confirmed the critical role of ferroptosis in NETs-induced ALI. These findings support a role for NETs-induced METTL3 modification and the subsequent induction of ferroptosis in the pathogenesis of sepsis-associated ALI.

PICK1 deficiency exacerbates sepsis-associated acute lung injury and impairs glutathione synthesis via reduction of xCT.

The role of oxidative stress has been well documented in the development of sepsis-induced acute lung injury (ALI). Protein interaction with C-kinase 1 (PICK1) participates in oxidative stress-related neuronal diseases. However, its function in lung infections and inflammatory diseases is not known. We therefore sought to investigate whether PICK1 is involved in sepsis-induced ALI. Cecal ligation and puncture (CLP) was performed in anesthetized wild type (WT) and PICK1 knock out (KO, PICK1-/-) mice with C57BL/6 background. At the time of CLP, mice were given fluid resuscitation. Mouse lungs were harvested at 24 and 72 h for Western blot analysis, qRT-PCR, BALF analysis, Hematoxylin and Eosin staining, TUNEL staining, maleimide staining, flow cytometry analysis, GCL, GSH, GSSG and cysteine levels measurement. A marked elevation of PICK1 mRNA and protein level were demonstrated in lung tissue, which was accompanied by increased production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) and consumption of glutathione (GSH). N-acetylcysteine (NAC), buthionine sulfoximine (BSO) and GSH-monoethyl ester (GSH-MEE) were injected into mice via caudal vein to regulate glutathione (GSH) level in lung. Alterations of lung GSH content induced PICK1 level change after CLP challenge. In PICK1-/- underwent with CLP, lung injury and survival were significantly aggravated compared with wild-type mice underwent with CLP. Concomitantly, CLP-induced lung cell apoptosis was exacerbated in PICK1-/- mice. The level of xCT, other than PKCα, in lung tissue was significantly lowered in PICK1-/- but not in wild type that underwent CLP surgery. Meanwhile, Nrf2 activation, which dominating xCT expression, was inhibited in PICK1-/- but not in wild type mice that underwent CLP surgery, as well. Moreover, higher level of PICK1 was detected in PBMCs of septic patients than healthy controls. Taken together, PICK1 plays a pivotal role in sepsis-induced ALI by regulating GSH synthesis via affecting the substrate-specific subunit of lung cystine/glutamate transporter, xCT.