Wnt5a-mediated autophagy contributes to the epithelial-mesenchymal transition of human bronchial epithelial cells during asthma.

BACKGROUND: The epithelial-mesenchymal transition (EMT) of human bronchial epithelial cells (HBECs) is essential for airway remodeling during asthma. Wnt5a has been implicated in various lung diseases, while its role in the EMT of HBECs during asthma is yet to be determined. This study sought to define whether Wnt5a initiated EMT, leading to airway remodeling through the induction of autophagy in HBECs. METHODS: Microarray analysis was used to investigate the expression change of WNT5A in asthma patients. In parallel, EMT models were induced using 16HBE cells by exposing them to house dust mites (HDM) or interleukin-4 (IL-4), and then the expression of Wnt5a was observed. Using in vitro gain- and loss-of-function approaches via Wnt5a mimic peptide FOXY5 and Wnt5a inhibitor BOX5, the alterations in the expression of the epithelial marker E-cadherin and the mesenchymal marker protein were observed. Mechanistically, the Ca2+/CaMKII signaling pathway and autophagy were evaluated. An autophagy inhibitor 3-MA was used to examine Wnt5a in the regulation of autophagy during EMT. Furthermore, we used a CaMKII inhibitor KN-93 to determine whether Wnt5a induced autophagy overactivation and EMT via the Ca2+/CaMKII signaling pathway. RESULTS: Asthma patients exhibited a significant increase in the gene expression of WNT5A compared to the healthy control. Upon HDM and IL-4 treatments, we observed that Wnt5a gene and protein expression levels were significantly increased in 16HBE cells. Interestingly, Wnt5a mimic peptide FOXY5 significantly inhibited E-cadherin and upregulated α-SMA, Collagen I, and autophagy marker proteins (Beclin1 and LC3-II). Rhodamine-phalloidin staining showed that FOXY5 resulted in a rearrangement of the cytoskeleton and an increase in the quantity of stress fibers in 16HBE cells. Importantly, blocking Wnt5a with BOX5 significantly inhibited autophagy and EMT induced by IL-4 in 16HBE cells. Mechanistically, autophagy inhibitor 3-MA and CaMKII inhibitor KN-93 reduced the EMT of 16HBE cells caused by FOXY5, as well as the increase in stress fibers, cell adhesion, and autophagy. CONCLUSION: This study illustrates a new link in the Wnt5a-Ca2+/CaMKII-autophagy axis to triggering airway remodeling. Our findings may provide novel strategies for the treatment of EMT-related diseases.

TREM-1 triggers necroptosis of macrophages through mTOR-dependent mitochondrial fission during acute lung injury.

BACKGROUND: Necroptosis of macrophages is a necessary element in reinforcing intrapulmonary inflammation during acute lung injury (ALI). However, the molecular mechanism that sparks macrophage necroptosis is still unclear. Triggering receptor expressed on myeloid cells-1 (TREM-1) is a pattern recognition receptor expressed broadly on monocytes/macrophages. The influence of TREM-1 on the destiny of macrophages in ALI requires further investigation. METHODS: TREM-1 decoy receptor LR12 was used to evaluate whether the TREM-1 activation induced necroptosis of macrophages in lipopolysaccharide (LPS)-induced ALI in mice. Then we used an agonist anti-TREM-1 Ab (Mab1187) to activate TREM-1 in vitro. Macrophages were treated with GSK872 (a RIPK3 inhibitor), Mdivi-1 (a DRP1 inhibitor), or Rapamycin (an mTOR inhibitor) to investigate whether TREM-1 could induce necroptosis in macrophages, and the mechanism of this process. RESULTS: We first observed that the blockade of TREM-1 attenuated alveolar macrophage (AlvMs) necroptosis in mice with LPS-induced ALI. In vitro, TREM-1 activation induced necroptosis of macrophages. mTOR has been previously linked to macrophage polarization and migration. We discovered that mTOR had a previously unrecognized function in modulating TREM-1-mediated mitochondrial fission, mitophagy, and necroptosis. Moreover, TREM-1 activation promoted DRP1Ser616 phosphorylation through mTOR signaling, which in turn caused surplus mitochondrial fission-mediated necroptosis of macrophages, consequently exacerbating ALI. CONCLUSION: In this study, we reported that TREM-1 acted as a necroptotic stimulus of AlvMs, fueling inflammation and aggravating ALI. We also provided compelling evidence suggesting that mTOR-dependent mitochondrial fission is the underpinning of TREM-1-triggered necroptosis and inflammation. Therefore, regulation of necroptosis by targeting TREM-1 may provide a new therapeutic target for ALI in the future.

The role of NOX4 in pulmonary diseases.

Nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) is a subtype of the NOX family, which is mainly expressed in the pulmonary vasculature and pulmonary endothelial cells in the respiratory system. NOX4 has unique characteristics, and is a constitutively active enzyme that primarily produces hydrogen peroxide. The signaling pathways associated with NOX4 are complicated. Negative and positive feedback play significant roles in regulating NOX4 expression. The role of NOX4 is controversial because NOX4 plays a protective or damaging role in different respiratory diseases. This review summarizes the structure, enzymatic properties, regulation, and signaling pathways of NOX4. This review then introduces the roles of NOX4 in different diseases in the respiratory system, such as acute respiratory distress syndrome, chronic obstructive pulmonary disease, and pulmonary fibrosis.

Epoxyeicosatrienoic acids inhibit the activation of NLRP3 inflammasome in murine macrophages.

Epoxyeicosatrienoic acids (EETs) derived from arachidonic acid exert anti-inflammation effects. We have reported that blocking the degradation of EETs with a soluble epoxide hydrolase (sEH) inhibitor protects mice from lipopolysaccharide (LPS)-induced acute lung injury (ALI). The underlying mechanisms remain essential questions. In this study, we investigated the effects of EETs on the activation of nucleotide-binding domain leucine-rich repeat-containing receptor, pyrin domain-containing-3 (NLRP3) inflammasome in murine macrophages. In an LPS-induced ALI murine model, we found that sEH inhibitor 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl), TPPU, profoundly attenuated the pathological injury and inhibited the activation of the NLRP3 inflammasome, characterized by the reduction of the protein expression of NLRP3, ASC, pro-caspase-1, interleukin precursor (pro-IL-1ß), and IL-1ß p17 in the lungs of LPS-treated mice. In vitro, primary peritoneal macrophages from C57BL/6 were primed with LPS and activated with exogenous adenosine triphosphate (ATP). TPPU treatment remarkably reduced the expression of NLRP3 inflammasome-related molecules and blocked the activation of NLRP3 inflammasome. Importantly, four EETs (5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET) inhibited the activation of NLRP3 inflammasome induced by LPS + ATP or LPS + nigericin in macrophages in various degree. While the inhibitory effect of 5,6-EET was the weakest. Mechanismly, EETs profoundly decreased the content of reactive oxygen species (ROS) and restored the calcium overload in macrophages receiving LPS + ATP stimulation. In conclusion, this study suggests that EETs inhibit the activation of the NLRP3 inflammasome by suppressing calcium overload and ROS production in macrophages, contributing to the therapeutic potency to ALI.

Inhibition of glycolysis alleviates lipopolysaccharide-induced acute lung injury in a mouse model.

Gluconic metabolic reprogramming, immune response, and inflammation are intimately linked. Glycolysis involves in the pathologic progress in acute and chronic inflammatory diseases. However, the involvement of glycolysis in the acute lung injury (ALI) is still unclear. This study investigated the role of glycolysis in an animal model of ALI. First, we found that lactate content in serum was remarkably increased in ALI patients and a murine model induced by intratracheal administration of lipopolysaccharide (LPS). The key proteins involving in glycolysis were robustly elevated, including HK2, PKM2, and HIF-1α. Intriguingly, inhibition of glycolysis by 2-deoxyglucose (2-DG) pronouncedly attenuated the lung tissue pathological injury, accumulation of neutrophil, oxidative stress, expression of proinflammatory factors in the lung of ALI mice induced by LPS. The 2-DG treatment also strongly suppressed the activation of the NOD-like receptor (NLR) family and pyrin domain-containing protein 3 (NLRP3) inflammasome. Furthermore, we investigated the role of glycolysis in the inflammatory response of primary murine macrophages activated by LPS in vitro. We found that the 2-DG treatment remarkably reduced the expression of proinflammatory factors induced by LPS, including tumor necrosis factor-α messenger RNA (mRNA), pro-interleukin (IL)-1ß mRNA, pro-IL-18 mRNA, NLRP3 mRNA, caspase-1 mRNA, and IL-1ß protein. Altogether, these data provide a novel link between gluconic metabolism reprogramming and uncontrolled inflammatory response in ALI. This study suggests glycolytic inhibition as an effective anti-inflammatory strategy in treating ALI.

mtDNA-cGAS-STING axis-dependent NLRP3 inflammasome activation contributes to postoperative cognitive dysfunction induced by sevoflurane in mice.

The activation of NLRP3 inflammasome in microglia is critical for neuroinflammation during postoperative cognitive dysfunction (POCD) induced by sevoflurane. However, the molecular mechanism by which sevoflurane activates the NLRP3 inflammasome in microglia remains unclear. The cGAS-STING pathway is an evolutionarily conserved inflammatory defense mechanism. The role of the cGAS-STING pathway in sevoflurane-induced NLRP3 inflammasome-dependent neuroinflammation and the underlying mechanisms require further investigation. We found that prolonged anesthesia with sevoflurane induced cognitive dysfunction and triggered the neuroinflammation characterized by the activation of NLRP3 inflammasome in vivo. Interestingly, the cGAS-STING pathway was activated in the hippocampus of mice receiving sevoflurane. While the blockade of cGAS with RU.521 attenuated cognitive dysfunction and NLRP3 inflammasome activation in mice. In vitro, we found that sevoflurane treatment significantly activated the cGAS-STING pathway in microglia, while RU.521 pre-treatment robustly inhibited sevoflurane-induced NLRP3 inflammasome activation. Mechanistically, sevoflurane-induced mitochondrial fission in microglia and released mitochondrial DNA (mtDNA) into the cytoplasm, which could be abolished with Mdivi-1. Blocking the mtDNA release via the mPTP-VDAC channel inhibitor attenuated sevoflurane-induced mtDNA cytosolic escape and reduced cGAS-STING pathway activation in microglia, finally inhibiting the NLRP3 inflammasome activation. Therefore, regulating neuroinflammation by targeting the cGAS-STING pathway may provide a novel therapeutic target for POCD.

TREM-1 governs NLRP3 inflammasome activation of macrophages by firing up glycolysis in acute lung injury.

The triggering receptor expressed on myeloid cells-1 (TREM-1) is a pro-inflammatory immune receptor potentiating acute lung injury (ALI). However, the mechanism of TREM-1-triggered inflammation response remains poorly understood. Here, we showed that TREM-1 blocking attenuated NOD-, LRR- and pyrin domain-containing 3 (NLRP3) inflammasome activation and glycolysis in LPS-induced ALI mice. Then, we observed that TREM-1 activation enhanced glucose consumption, induced glycolysis, and inhibited oxidative phosphorylation in macrophages. Specifically, inhibition of glycolysis with 2-deoxyglucose diminished NLRP3 inflammasome activation of macrophages triggered by TREM-1. Hypoxia-inducible factor-1α (HIF-1α) is a critical transcriptional regulator of glycolysis. We further found that TREM-1 activation facilitated HIF-1α accumulation and translocation to the nucleus via the phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway. Inhibiting mTOR or HIF-1α also suppressed TREM-1-induced metabolic reprogramming and NLRP3/caspase-1 activation. Overall, the mTOR/HIF-1α/glycolysis pathway is a novel mechanism underlying TREM-1-governed NLRP3 inflammasome activation. Therapeutic targeting of the mTOR/HIF-1α/glycolysis pathway in TREM-1-activated macrophages could be beneficial for treating or preventing inflammatory diseases, such as ALI.

Epoxyeicosatrienoic acids alleviate alveolar epithelial cell senescence by inhibiting mitophagy through NOX4/Nrf2 pathway.

Alveolar epithelial cell (AEC) senescence is considered to be a universal pathological feature of many chronic pulmonary diseases. Our previous study found that epoxyeicosatrienoic acids (EETs), produced from arachidonic acid (ARA) through the cytochrome P450 cyclooxygenase (CYP) pathway, have significant negative regulatory effects on cellular senescence in AECs. However, the exact mechanisms by which EETs alleviate the senescence of AECs still need to be further explored. In the present study, we observed that bleomycin (BLM) induced enhanced mitophagy accompanied by increased mitochondrial ROS (mito-ROS) content in the murine alveolar epithelial cell line MLE12. While EETs reduced BLM-induced mitophagy and mito-ROS content in MLE12 cells, and the mechanism was related to the regulation of NOX4/Nrf2-mediated redox imbalance. Furthermore, we found that inhibition of EETs degradation could significantly inhibit mitophagy and regulate NOX4/Nrf2 balance to exert anti-oxidant effects in D-galactose-induced premature aging mice. Collectively, these findings may provide new ideas for treating age-related pulmonary diseases by targeting EETs to improve mitochondrial dysfunction and reduce oxidative stress.

TREM-1 exacerbates bleomycin-induced pulmonary fibrosis by aggravating alveolar epithelial cell senescence in mice.

Our previous study showed that triggering receptors expressed on myeloid cell-1 (TREM-1) was upregulated in bleomycin (BLM)-induced pulmonary fibrosis (PF) mouse model. However, the role of TREM-1 in the development of PF and its underlying mechanism remain unclear. Herein, we report that the prophylactical blockade of TREM-1 using a decoy peptide dodecapeptide (LR12) exerted protective effects against BLM-induced PF in mice, with a higher survival rate, attenuated tissue injury, and less extracellular matrix deposition. Interestingly, therapeutic blockade of TREM-1 at the early stage of fibrosis also attenuated BLM-induced PF, suggesting a non-inflammatory effect. More importantly, we observed that TREM-1 blockade with LR12 significantly reduced the expression of the senescence-relative protein, including p16, p21, p53, and γ-H2AX in the lungs of PF mice. Notably, TREM-1 was upregulated in alveolar epithelial cells (AECs) and correlated with the levels of senescence markers in BLM-treated mice. In vitro, activating TREM-1 with an agonistic antibody exacerbated BLM-induced senescence in MLE12 cells, a murine AEC cell line. Furthermore, prophylactic or therapeutic blockade of TREM-1 protected MLE12 cells from senescence induced by BLM or H2O2. In conclusion, our findings elucidate a pro-fibrotic effect of TREM-1 by inducing AECs senescence in PF, providing a potential strategy for fibrotic disease treatment.

Mitochondrial citrate accumulation drives alveolar epithelial cell necroptosis in lipopolysaccharide-induced acute lung injury.

Necroptosis is the major cause of death in alveolar epithelial cells (AECs) during acute lung injury (ALI). Here, we report a previously unrecognized mechanism for necroptosis. We found an accumulation of mitochondrial citrate (citratemt) in lipopolysaccharide (LPS)-treated AECs because of the downregulation of Idh3α and citrate carrier (CIC, also known as Slc25a1). shRNA- or inhibitor-mediated inhibition of Idh3α and Slc25a1 induced citratemt accumulation and necroptosis in vitro. Mice with AEC-specific Idh3α and Slc25a1 deficiency exhibited exacerbated lung injury and AEC necroptosis. Interestingly, the overexpression of Idh3α and Slc25a1 decreased citratemt levels and rescued AECs from necroptosis. Mechanistically, citratemt accumulation induced mitochondrial fission and excessive mitophagy in AECs. Furthermore, citratemt directly interacted with FUN14 domain-containing protein 1 (FUNDC1) and promoted the interaction of FUNDC1 with dynamin-related protein 1 (DRP1), leading to excessive mitophagy-mediated necroptosis and thereby initiating and promoting ALI. Importantly, necroptosis induced by citratemt accumulation was inhibited in FUNDC1-knockout AECs. We show that citratemt accumulation is a novel target for protection against ALI involving necroptosis.

Activation of NLRP3 inflammasome up-regulates TREM-1 expression in murine macrophages via HMGB1 and IL-18.

NOD-, LRR- and pyrin domain-containing 3 (NLRP3) inflammasome and triggering receptor expressed on myeloid cells-1 (TREM-1) are considered critical orchestrators of the inflammatory response in acute lung injury (ALI). However, few assumptions are based on the relationship between them. Here, we investigated the effect of NLRP3 inflammasome activation on the TREM-1 expression in lipopolysaccharide (LPS)-induced ALI and macrophages. We found that inhibition of the NLRP3 inflammasome reduced the TREM-1 expression and pathological lung injury in mice with ALI. Then, primary murine macrophages were used to dissect the underlying mechanistic events of the activation NLRP3 inflammasome involved in the TREM-1 expression. Our results demonstrated that the conditioned medium (CM) from NLRP3 inflammasome-activated-macrophages up-regulated the TREM-1 expression in macrophages, while this effect was reversed by an NLRP3 inflammasome inhibitor MCC950. Furthermore, neutralizing antibodies anti-IL-18 and anti-HMGB1 reduced the TREM-1 expression induced by NLRP3 inflammasome activation. Mechanistically, we found that CM from NLRP3 inflammasome-activated-macrophages increased the level of inhibitor κB kinase protein phosphorylation (p-IκBα) and reactive oxygen species (ROS) content, and promoted IκBα protein degradation in macrophages. While the inhibition of nuclear factor kappa-B (NF-κB) and scavenging ROS eliminated the up-regulation of TREM-1 induced by the NLRP3 inflammasome activation in macrophages. In summary, our study confers NLRP3 inflammasome as a new trigger of TREM-1 signing, which allows additional insight into the pathological of the inflammatory response in ALI.

A COX-2/sEH dual inhibitor PTUPB ameliorates cecal ligation and puncture-induced sepsis in mice via anti-inflammation and anti-oxidative stress.

Arachidonic acid can be metabolized to prostaglandins and epoxyeicosatrienoic acids (EETs) by cyclooxygenase-2 (COX-2) and cytochrome P450 (CYP), respectively. While protective EETs are degraded by soluble epoxide hydrolase (sEH) very fast. We have reported that dual inhibition of COX-2 and sEH with specific inhibitor PTUPB shows anti-pulmonary fibrosis and renal protection. However, the effect of PTUPB on cecal ligation and puncture (CLP)-induced sepsis remains unclear. The current study aimed to investigate the protective effects of PTUPB against CLP-induced sepsis in mice and the underlying mechanisms. We found that COX-2 expressions were increased, while CYPs expressions were decreased in the liver, lung, and kidney of mice undergone CLP. PTUPB treatment significantly improved the survival rate, reduced the clinical scores and systemic inflammatory response, alleviated liver and kidney dysfunction, and ameliorated the multiple-organ injury of the mice with sepsis. Besides, PTUPB treatment reduced the expression of hypoxia-inducible factor-1α in the liver, lung, and kidney of septic mice. Importantly, we found that PTUPB treatment suppressed the activation of NLRP3 inflammasome in the liver and lung of septic mice. Meanwhile, we found that PTUPB attenuated the oxidative stress, which contributed to the activation of NLRP3 inflammasome. Altogether, our data, for the first time, demonstrate that dual inhibition of COX-2 and sEH with PTUPB ameliorates the multiple organ dysfunction in septic mice.

A COX-2/sEH dual inhibitor PTUPB alleviates lipopolysaccharide-induced acute lung injury in mice by inhibiting NLRP3 inflammasome activation.

Rationale: Dysregulation of arachidonic acid (ARA) metabolism results in inflammation; however, its role in acute lung injury (ALI) remains elusive. In this study, we addressed the role of dysregulated ARA metabolism in cytochromes P450 (CYPs) /cyclooxygenase-2 (COX-2) pathways in the pathogenesis of lipopolysaccharide (LPS)-induced ALI in mice. Methods: The metabolism of CYPs/COX-2-derived ARA in the lungs of LPS-induced ALI was investigated in C57BL/6 mice. The COX-2/sEH dual inhibitor PTUPB was used to establish the function of CYPs/COX-2 dysregulation in ALI. Primary murine macrophages were used to evaluate the underlying mechanism of PTUPB involved in the activation of NLRP3 inflammasome in vitro. Results: Dysregulation of CYPs/COX-2 metabolism of ARA occurred in the lungs and in primary macrophages under the LPS challenge. Decrease mRNA expression of Cyp2j9, Cyp2j6, and Cyp2j5 was observed, which metabolize ARA into epoxyeicosatrienoic acids (EETs). The expressions of COX-2 and soluble epoxide hydrolase (sEH), on the other hand, was significantly upregulated. Pre-treatment with the dual COX-2 and sEH inhibitor, PTUPB, attenuated the pathological injury of lung tissues and reduced the infiltration of inflammatory cells. Furthermore, PTUPB decreased the pro-inflammatory factors, oxidative stress, and activation of NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome in LPS-induced ALI mice. PTUPB pre-treatment remarkably reduced the activation of macrophages and NLRP3 inflammasome in vitro. Significantly, both preventive and therapeutic treatment with PTUPB improved the survival rate of mice receiving a lethal dose of LPS. Conclusion: The dysregulation of CYPs/COX-2 metabolized ARA contributes to the uncontrolled inflammatory response in ALI. The dual COX-2 and sEH inhibitor PTUPB exerts anti-inflammatory effects in treating ALI by inhibiting the NLRP3 inflammasome activation.

Exogenous angiotensin (1-7) directly inhibits epithelial-mesenchymal transformation induced by transforming growth factor-ß1 in alveolar epithelial cells.

Accumulating evidence indicates that angiotensin (1-7) [Ang-(1-7)] protects against idiopathic pulmonary fibrosis (IPF) in animal experiments. However, whether Ang-(1-7) effectively inhibits epithelial-mesenchymal transition (EMT) induced by transforming growth factor-ß1 (TGF-ß1) remains unclear. The aim of this study is to examine the eff ;ects of Ang-(1-7) on TGF-ß1-induced EMT in human alveolar epithelial cells. We found that angiotensin-converting enzyme 2 (ACE2) /Ang-(1-7)/MasR were decreased in the lungs of mice with IPF induced by bleomycin, and were negatively correlated with Tgfb1 mRNA expression. In vitro, our data showed that exogenous Ang-(1-7) restored the expression of E-cadherin and decreased the expressions of α-SMA and Vimentin induced by TGF-ß1 in A549 cells. Ang-(1-7) also reduced TGF-ß1-induced migration and synthesis of the extracellular matrix, such as collagen Ⅰ and collagen Ⅲ. Mechanistically, we observed that Ang-(1-7) directly inhibited TGF-ß1-induced phosphorylation of Smad2 and Smad3, and suppressed the expression of the downstream target gene of TGF-ß1-Smad signaling, including ZEB1, ZEB2, TWIST, and SNAIL1. Additionally, phosphorylation of mTOR induced by TGF-ß1 also been suppressed by Ang-(1-7) treatment in A549 cells. Interestingly, we found that TGF-ß1 strongly suppressed the expression of ACE2 in A549 cells through inhibiting SIRT1. In conclusion, our findings indicate that Ang-(1-7) directly inhibits TGF-ß1-induced EMT in alveolar epithelial cells via disruption of TGF-ß1-Smad signaling pathway, contributing to the protective effect against IPF.