Resolvin D1 attenuates mechanical stretch-induced pulmonary fibrosis via epithelial-mesenchymal transition.

Mechanical ventilation-induced pulmonary fibrosis plays an important role in the high mortality rate of acute respiratory distress syndrome (ARDS). Resolvin D1 (RvD1) displays potent proresolving activities. Epithelial-mesenchymal transition (EMT) has been proved to be an important pathological feature of lung fibrosis. This study aimed to investigate whether RvD1 can attenuate mechanical ventilation-induced lung fibrosis. Human lung epithelial (BEAS-2B) cells were pretreated with RvD1 for 30 min and exposed to acid for 10 min before being subjected to mechanical stretch for 48 h. C57BL/6 mice were subjected to intratracheal acid aspiration followed by mechanical ventilation 24 h later (peak inspiratory pressure 22 cmH2O, positive end-expiratory pressure 2 cmH2O, and respiratory rate 120 breaths/min for 2 h). RvD1 was injected into mice for 5 consecutive days after mechanical ventilation. Treatment with RvD1 significantly inhibited mechanical stretch-induced mesenchymal markers (vimentin and α-smooth muscle actin) and stimulated epithelial markers (E-cadherin). Tert-butyloxycarbonyl 2 (BOC-2), a lipoxin A4 receptor/formyl peptide receptor 2 (ALX/FPR2) antagonist, is known to inhibit ALX/FPR2 function. BOC-2 could reverse the beneficial effects of RvD1. The antifibrotic effect of RvD1 was associated with the suppression of Smad2/3 phosphorylation. This study demonstrated that mechanical stretch could induce EMT and pulmonary fibrosis and that treatment with RvD1 could attenuate mechanical ventilation-induced lung fibrosis, thus highlighting RvD1 as an effective therapeutic agent against pulmonary fibrosis associated with mechanical ventilation.

Activation of death-associated protein kinase 1 promotes neutrophil apoptosis to accelerate inflammatory resolution in acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is a uniform progression of overwhelming inflammation in lung tissue with extensive infiltration of inflammatory cells. Neutrophil apoptosis is thought to be a significant process in the control of the resolution phase of inflammation. It has been proved that 5-Aza-2'-deoxycytidine (Aza) can inhibit cancer by activating death-associated protein kinase 1 (DAPK1) to promote apoptosis. However, the effect of DAPK1 on neutrophil apoptosis is unclear, and research on the role of Aza in inflammation is lacking. Here, we investigated whether Aza can regulate DAPK1 expression to influence the fate of neutrophils in ARDS. In vitro, we stimulated neutrophil-like HL-60 (dHL-60) cells with different concentrations of Aza for different durations and used RNA interference to up- or downregulate DAPK1 expression. We observed that culturing dHL-60 cells with Aza increased apoptosis by inhibiting NF-κB activation to modulate the expression of Bcl-2 family proteins, which was closely related to the levels of DAPK1. In vivo, ARDS was evoked by intratracheal instillation of lipopolysaccharide (LPS; 3 mg/kg). One hour after LPS administration, mice were treated with Aza (1 mg/kg, i.p.). To inhibit DAPK1 expression, mice were intraperitoneally injected with a DAPK1 inhibitor. Aza treatment accelerated inflammatory resolution in LPS-induced ARDS by suppressing pulmonary edema, alleviating lung injury and decreasing the infiltration of inflammatory cells in bronchoalveolar lavage fluid (BALF). Moreover, Aza reduced the production of proinflammatory cytokines. However, administration of the DAPK1 inhibitor attenuated the protective effects of Aza. Similarly, the proapoptotic function of Aza was prevented when DAPK1 was inhibited either in vivo or in vitro. In summary, Aza promotes neutrophil apoptosis by activating DAPK1 to accelerate inflammatory resolution in LPS-induced ARDS. This study provides the first evidence that Aza prevents LPS-induced neutrophil survival by modulating DAPK1 expression.

Lipocalin 10 is essential for protection against inflammation-triggered vascular leakage by activating LDL receptor-related protein 2-slingshot homologue 1 signalling pathway.

AIMS: Systemic inflammation occurs commonly during many human disease settings and increases vascular permeability, leading to organ failure, and lethal outcomes. Lipocalin 10 (Lcn10), a poorly characterized member of the lipocalin family, is remarkably altered in the cardiovascular system of human patients with inflammatory conditions. Nonetheless, whether Lcn10 regulates inflammation-induced endothelial permeability remains unknown. METHODS AND RESULTS: Systemic inflammation models were induced using mice by injection of endotoxin lipopolysaccharide (LPS) or caecal ligation and puncture (CLP) surgery. We observed that the expression of Lcn10 was dynamically altered only in endothelial cells (ECs), but not in either fibroblasts or cardiomyocytes isolated from mouse hearts following the LPS challenge or CLP surgery. Using in vitro gain- and loss-of-function approaches and an in vivo global knockout mouse model, we discovered that Lcn10 negatively regulated endothelial permeability upon inflammatory stimuli. Loss of Lcn10 augmented vascular leakage, leading to severe organ damage and higher mortality following LPS challenge, compared to wild-type controls. By contrast, overexpression of Lcn10 in ECs displayed opposite effects. A mechanistic analysis revealed that both endogenous and exogenous elevation of Lcn10 in ECs could activate slingshot homologue 1 (Ssh1)-Cofilin signalling cascade, a key axis known to control actin filament dynamics. Accordingly, a reduced formation of stress fibre and increased generation of cortical actin band were exhibited in Lcn10-ECs, when compared to controls upon endotoxin insults. Furthermore, we identified that Lcn10 interacted with LDL receptor-related protein 2 (LRP2) in ECs, which acted as an upstream factor of the Ssh1-Confilin signalling. Finally, injection of recombinant Lcn10 protein into endotoxic mice showed therapeutic effects against inflammation-induced vascular leakage. CONCLUSION: This study identifies Lcn10 as a novel regulator of EC function and illustrates a new link in the Lcn10-LRP2-Ssh1 axis to controlling endothelial barrier integrity. Our findings may provide novel strategies for the treatment of inflammation-related diseases.

Screening of potential key ferroptosis-related genes in sepsis.

Background: Sepsis leads to multiple organ dysfunction caused by a dysregulated host response to infection with a high incidence and mortality. The effect of ferroptosis on the development of sepsis remains unclear. In this study, we aimed to identify the key ferroptosis-related genes involved in sepsis and further explore the potential biological functions of these ferroptosis-related genes in sepsis using bioinformatics analysis. Methods: The GSE13904 (from children) and GSE28750 (from adults) datasets were downloaded from the Gene Expression Omnibus (GEO). The ferroptosis-related genes were obtained from the FerrDb database. The ferroptosis-related differentially expressed genes (DEGs) were screened by the limma R package. The DAVID online database or clusterProfiler R package was used for the functional enrichment analysis. Then, the STRING database was used to predict the interactions of proteins, and the CytoHubba plugin of Cytoscape was used to confirm key clustering modules. Then, the miRNAs and lncRNAs associated with the key clustering modules were predicted by miRWalk 2.0 and LncBase v.2 respectively. Finally, we generated a cecal ligation and puncture (CLP) polymicrobial sepsis model in C57 male mice and examined the expression of the mRNAs and noncoding RNAs of interest in peripheral blood leukocytes by PCR during the acute inflammation phase. Results: In total, 34 ferroptosis-related DEGs were identified in both adult and pediatric septic patients. These ferroptosis-related DEGs were mainly enriched in inflammatory pathways. Then, a significant clustering module containing eight genes was identified. Among them, the following five genes were closely associated with the MAPK signaling pathway: MAPK14, MAPK8, DUSP1, MAP3K5 and MAPK1. Then, crucial miRNAs and lncRNAs associated with biomarker MAPK-related genes were also identified. In particular, let-7b-5p and NEAT1 were selected as noncoding RNAs of interest because of their correlation with ferroptosis in previous studies. Finally, we examined the mRNAs, miRNAs and lncRNAs of interest using CLP-induced sepsis in peripheral blood leukocytes of mice. The results showed that MAPK14, MAPK8, MAP3K5, MAPK1 and NEAT1 were upregulated, while DUSP1 and let-7b-5p were downregulated in the CLP group compared with the sham group. Conclusions: The MAPK signaling pathway may play a key role in regulating ferroptosis during sepsis. This study provides a valuable resource for future studies investigating the mechanism of MAPK-related ferroptosis in sepsis.

Immunopathological Roles of Neutrophils in Virus Infection and COVID-19.

ABSTRACT: Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been spread around the world and is currently affecting global public health. Clinical evidence indicates that the elevated number of peripheral neutrophils and higher ratio of neutrophils-to-lymphocytes are correlated with severe outcomes in COVID-19 patients, suggesting the possible immunopathological role of neutrophils during SARS-CoV-2 infection. As an abundant innate immune cell type, neutrophils are well known for their contributions to antimicrobial defense. However, their dysfunction is also associated with different inflammatory signatures during the pathogenesis of infection. Herein, in this mini-review, we summarize the recent progress on the potential role of neutrophils during COVID-19-associated inflammatory responses. In particular, we highlight the interactions between neutrophils and viruses as well as the relationship of neutrophils with cytokine storm and thrombosis in COVID-19 patients. Lastly, we discuss the importance of neutrophils as potential therapeutic targets for COVID-19.

Administration of GDF3 Into Septic Mice Improves Survival via Enhancing LXRα-Mediated Macrophage Phagocytosis.

The defective eradication of invading pathogens is a major cause of death in sepsis. As professional phagocytic cells, macrophages actively engulf/kill microorganisms and play essential roles in innate immune response against pathogens. Growth differentiation factor 3 (GDF3) was previously implicated as an important modulator of inflammatory response upon acute sterile injury. In this study, administration of recombinant GDF3 protein (rGDF3) either before or after CLP surgery remarkably improved mouse survival, along with significant reductions in bacterial load, plasma pro-inflammatory cytokine levels, and organ damage. Notably, our in vitro experiments revealed that rGDF3 treatment substantially promoted macrophage phagocytosis and intracellular killing of bacteria in a dose-dependent manner. Mechanistically, RNA-seq analysis results showed that CD5L, known to be regulated by liver X receptor α (LXRα), was the most significantly upregulated gene in rGDF3-treated macrophages. Furthermore, we observed that rGDF3 could promote LXRα nuclear translocation and thereby, augmented phagocytosis activity in macrophages, which was similar as LXRα agonist GW3965 did. By contrast, pre-treating macrophages with LXRα antagonist GSK2033 abolished beneficial effects of rGDF3 in macrophages. In addition, rGDF3 treatment failed to enhance bacteria uptake and killing in LXRα-knockout (KO) macrophages. Taken together, these results uncover that GDF3 may represent a novel mediator for controlling bacterial infection.

NecroX-5 alleviate lipopolysaccharide-induced acute respiratory distress syndrome by inhibiting TXNIP/NLRP3 and NF-κB.

The activation of NLRP3 inflammasome and NF-κB pathway, associating with oxidativestress, have been implicated in the development of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). NecroX-5 has been reported to exhibit theeffectsofanti-oxidation and anti-stress in various diseases. However, the role of NecroX-5 in ALI has not been explicitly demonstrated. The aim of this study was to explore the therapeutic effects and potential mechanism action of NecroX-5 on ALI. Here, we found that NecroX-5 pretreatment dramatically diminished the levels of IL-1ß, IL-18 and ROS in in RAW264.7 cells challenged with LPS and ATP. Furthermore, NecroX-5 suppressed the activation of NLRP3 inflammasome and NF-κB signalpathway. In addition, NecroX-5 also inhibited the thioredoxin-interacting protein (TXNIP) expression. In vivo, NecroX-5 reduced the LPS-induced lung histopathological injury, the number of TUNEL-positive cells, lung wet/dry (W/D) ratio, levels of total protein and inflammatory cytokines in the bronchoalveolar lavage fluid (BALF) in mice. Additionally, LPS-induced upregulation of myeloperoxidase (MPO), ROS production and malondialdehyde (MDA) were inhibited by NecroX-5 administration. Thus, our results demonstrate that NecroX-5 protects against LPS-induced ALI by inhibiting TXNIP/NLRP3 and NF-κB.

GDF3 Protects Mice against Sepsis-Induced Cardiac Dysfunction and Mortality by Suppression of Macrophage Pro-Inflammatory Phenotype.

Macrophages are critical for regulation of inflammatory response during endotoxemia and septic shock. However, the mediators underlying their regulatory function remain obscure. Growth differentiation factor 3 (GDF3), a member of transforming growth factor beta (TGF-ß) superfamily, has been implicated in inflammatory response. Nonetheless, the role of GDF3 in macrophage-regulated endotoxemia/sepsis is unknown. Here, we show that serum GDF3 levels in septic patients are elevated and strongly correlate with severity of sepsis and 28-day mortality. Interestingly, macrophages treated with recombinant GDF3 protein (rGDF3) exhibit greatly reduced production of pro-inflammatory cytokines, comparing to controls upon endotoxin challenge. Moreover, acute administration of rGDF3 to endotoxin-treated mice suppresses macrophage infiltration to the heart, attenuates systemic and cardiac inflammation with less pro-inflammatory macrophages (M1) and more anti-inflammatory macrophages (M2), as well as prolongs mouse survival. Mechanistically, GDF3 is able to activate Smad2/Smad3 phosphorylation, and consequently inhibits the expression of nod-like receptor protein-3 (NLRP3) in macrophages. Accordingly, blockade of Smad2/Smad3 phosphorylation with SB431542 significantly offsets rGDF3-mediated anti-inflammatory effects. Taken together, this study uncovers that GDF3, as a novel sepsis-associated factor, may have a dual role in the pathophysiology of sepsis. Acute administration of rGDF3 into endotoxic shock mice could increase survival outcome and improve cardiac function through anti-inflammatory response by suppression of M1 macrophage phenotype. However, constitutive high levels of GDF3 in human sepsis patients are associated with lethality, suggesting that GDF3 may promote macrophage polarization toward M2 phenotype which could lead to immunosuppression.

BML-111 accelerates the resolution of inflammation by modulating the Nrf2/HO-1 and NF-κB pathways in rats with ventilator-induced lung injury.

The timely resolution of pulmonary inflammation coordinated by endogenous pro-resolving mediators helps limit lung tissue injury, but few endogenous pro-resolving mediators that are normally operative during acute inflammation. The protective effects of BML-111 (5(S)-6(R)-7-trihydroxyheptanoic acid methyl ester), a potent commercially available anti-inflammatory and pro-resolving mediator, on ventilation-induced lung injury (VILI) have been extensively studied, but its characteristics as a pro-resolving mediator have not. Here, anesthetized Sprague-Dawley rats were ventilated with a high tidal volume (20 mL/kg, HVT) for 1 h and randomly allocated to recover for 6, 12, 24, 48, 72, 96 or 168 h; BML-111 was administered at the peak of inflammation to evaluate its pro-resolving effect on VILI. The one-hour HVT induced a maximal pulmonary inflammatory response at 12 h that was largely resolved by 72 h. BML-111 largely resolved the maximal inflammatory response at 48 h; the resolution interval (Ri) was shortened by 26 h. Similarly, HVT elicited a time course of changes in histopathology and pulmonary edema, and BML-111 alleviates these changes. Mechanistically, neutrophil apoptosis was significantly increased in BML-111-treated rats subjected to HVT. The apoptosis inhibitor z-VAD-fmk partially reversed the proapoptotic actions of BML-111 on neutrophil and the resolving effects of BML-111 on VILI but had no effect alone. Importantly, the HVT treatment activated the nuclear factor E2-related factor 2(Nrf2)/heme oxygenase-1(HO-1) and NF-κB signaling pathways in the lung tissue, and BML-111 further induced Nrf2 and HO-1 expression but inhibited the NF-κB pathway. Intriguingly, when we inhibited the Nrf2/HO-1 pathway with the HO-1 inhibitor zinc protoporphyrin IX (ZnPPIX), Nrf2 expression was further increased, but the inhibitory effects of BML-111 on the NF-κB pathway and on the subsequent inflammatory response, and the proapoptotic actions on neutrophil were reversed. The results suggest that BML-111 promotes the resolution of HVT-induced inflammation to mitigate VILI in rats, perhaps by modulating the Nrf2/HO-1 and NF-κB pathways and subsequently increasing neutrophil apoptosis.

Trichostatin A modulates the macrophage phenotype by enhancing autophagy to reduce inflammation during polymicrobial sepsis.

Sepsis is a syndrome of life-threatening organ dysfunction caused by dysregulated host responses to infection. Macrophage polarization is a key process involved in the pathogenesis of sepsis. Recent evidence has demonstrated that autophagy participates in the regulation of macrophage polarization in different phases of inflammation. Here, we investigated whether trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, promotes the macrophage M2 phenotype by enhancing autophagy to counteract excessive inflammation in a cecal ligation and puncture (CLP) mouse model. TSA stimulation increased the proportions of M2 marker (CD206, CD124 and CD23)-labeled RAW264.7 macrophages. Furthermore, with increasing TSA doses, autophagy was enhanced gradually. Interestingly, the autophagy activator rapamycin (Rap), also known as an mTOR inhibitor, unexpectedly decreased the proportions of M2 marker-labeled macrophages. However, TSA treatment reversed the Rap-induced decreases in CD206-labeled macrophages. Next, we stimulated different groups of RAW264.7 cells with the autophagy inhibitors MHY1485 or 3-methyladenine (3-MA). Inhibition of autophagy at any stage in the process suppressed TSA-induced macrophage M2 polarization, but the effect was not associated with mTOR activity. In vivo, TSA administration promoted peritoneal macrophage M2 polarization, increased LC3 II expression, attenuated sepsis-induced organ (lung, liver and kidney) injury, and altered systemic inflammatory cytokine secretion. However, 3-MA abolished the protective effects of TSA in CLP mice and decreased the number of M2 peritoneal macrophages. Therefore, TSA promotes the macrophage M2 phenotype by enhancing autophagy to reduce systemic inflammation and ultimately improves the survival of mice with polymicrobial sepsis.

[Mechanism of resolvins in reducing the inflammation reaction in inflammatory diseases].

OBJECTIVE: Inflammation is an important mechanism for the host to resist external infection and injury. Under normal conditions, adaptive inflammatory response not only is conducive to the repair of damaged cells and tissues, but plays an important role to promote the internal environment to restored homeostasis. However, when the immune function is damaged, the unresolved and uncontrolled inflammation will cause non-specific tissue damage, which is linked to a variety of inflammatory diseases. In the acute inflammation, resolvins, a group of endogenous small molecular lipid mediators which are biosynthesized by polyunsaturated fatty acids, possess anti-inflammatory and pro-resolving actions, which are benefit for the repair of damaged tissue. Therefore, it is helpful to deeply explore the mechanisms of resolvins in inflammation-resolution phases of acute lung injury (ALI) and sepsis, which are favor of understanding the complex processes and provide new treatment for the inflammatory diseases thereby reducing the mortality in the intensive care unit (ICU).

Protectin DX increases survival in a mouse model of sepsis by ameliorating inflammation and modulating macrophage phenotype.

Recently, a serial of studies have demonstrated that lipid mediators derived from Omega-3 fatty acid docosahexaenoic acid have pro-resolving or anti-inflammatory effects in many inflammatory diseases. Here, we sought to evaluate whether Protectin DX (PDX, an isomer of Protecin D1), a newly identified lipid mediator, could protect mice against sepsis and explore the underling mechanism. Animal model of sepsis was established by cecum ligation and puncture (CLP). We found that PDX increased overall survival rate within eight days and attenuated multiple organ injury in septic mice. In addition, PDX reduced pro-inflammatory cytokines and bacterial load 24 h after CLP. Moreover, PDX promoted phagocytosis of peritoneal macrophages and increased the percentage of M2 macrophages in peritoneum of septic mice. In vitro, M2 macrophage markers (Arg1 and Ym1) and its transcriptional regulator (peroxisome proliferator-activated receptor-γ, PPAR-γ) were upregulated in Raw264.7 macrophages challenged with PDX. GW9662 (a PPAR-γ inhibitor) and PPAR-γ siRNA abrogated the induction of Arg1 and Ym1 by PDX in Raw264.7 cells. Taken together, our results suggest that PDX is able to promote M2 polarization, enhance phagocytosis activity of macrophage and accelerate resolution of inflammation, finally leading to increased survival rate of septic mice.