Xiaoli Su

Neutrophil extracellular traps are indirectly triggered by lipopolysaccharide and contribute to acute lung injury.

Neutrophil extracellular traps (NETs) facilitate the extracellular killing of pathogens. However, excessive NETs formation and poor degradation are associated with exacerbated immune responses and tissue injury. In this study, we investigated the role of NETs in lipopolysaccharide (LPS)-mediated acute lung injury (ALI) and assessed the use of DNase I, for the treatment of ALI. Additionally, we focused on the controversial issue of whether LPS directly induces NETs release in vitro. NETs formation was detected in murine ALI tissue in vivo and was associated with increased NETs markers, citrullinated-histone H3 tissue levels and NET-DNA levels in BALF. Treatment with DNase I significantly degraded NETs and reduced citrullinated-histone H3 levels, which protected against ALI and ameliorated pulmonary oedema and total protein in BALF. In addition, DNase I significantly reduced IL-6 and TNF-α levels in plasma and BALF. In vitro, LPS-activated platelets rather than LPS alone efficiently induced NETs release. In conclusion, NETs formed during LPS-induced ALI, caused organ damage and initiated the inflammatory response. NETs degradation by DNase I promoted NET-protein clearance and protected against ALI in mice; thus, DNase I may be a new potential adjuvant for ALI therapy. Specifically, LPS induced NETs formation in an indirect manner via platelets activation.

Interferon Regulatory Factor-1 Mediates Alveolar Macrophage Pyroptosis During LPS-Induced Acute Lung Injury in Mice.

ABSTRACT Previously, we demonstrated that pyroptosis in alveolar macrophages (AMs) plays an essential role in lipopolysaccharide (LPS)-induced acute lung injury. However, the underlying mechanism remains largely unclear. Here, we show that the absence of interferon regulatory factor 1 (IRF-1) in genetic knock-out mice strongly abrogates pyroptosis in AMs and alleviates the LPS-induced lung injury and systemic inflammation. Our study demonstrates that IRF-1 contributes to caspase-1 activation and apoptosis-associated speck-like protein containing a caspase activation and recruitment domain pyroptosome formation in AMs and leads to downstream inflammatory cytokine release, including that of IL-1&bgr;, IL-18, and HMGB1. The nuclear translocation of IRF-1 is linked to the presence of toll-like receptor 4 (TLR4). Our findings suggest that pyroptosis and the downstream inflammatory response in AMs induced by LPS is a process that is dependent on TLR4-mediated up-regulation of IRF-1. In summary, IRF-1 plays a key role in controlling caspase-1-dependent pyroptosis and inflammation.

Inhibition of Alveolar Macrophage Pyroptosis Reduces Lipopolysaccharide-induced Acute Lung Injury in Mice.

Background:Pyroptosis is the term for caspase-1-dependent cell death associated with pro-inflammatory cytokines. The role of alveolar macrophage (AM) pyroptosis in the pathogenesis of the acute lung injury and acute respiratory distress syndrome (ALI/ARDS) remains unclear. Methods:C57BL/6 wild-type mice were assigned to sham, lipopolysaccharide (LPS) + vehicle, LPS + acetyl-tyrosyl-valyl- alanyl-aspartyl-chloromethylketone (Ac-YVAD-CMK) and LPS + Z-Asp-Glu-Val-Asp-fluoromethylketone groups. Mice were given intraperitoneal (IP) injections of LPS. Drugs were IP injected 1 h before LPS administration. Mice were sacrificed 16 h after LPS administration, and AMs were isolated. Western blot analysis for active caspase-1 and cleaved caspase-3, evaluation of lung injury and a cytokine release analysis were performed. AMs were treated with LPS and adenosine triphosphate (ATP); caspase-1-dependent cell death was evaluated using flow cytometry; the apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) pyroptosomes were examined by immunofluorescence. Results:The expression of activated caspase-1 in AMs was enhanced following LPS challenge compared with the sham group. In the ex vivo study, the caspase-1/propidium iodide-positive cells, caspase-1 specks and ASC pyroptosomes were up-regulated in AMs following LPS/ATP stimulation. The specific caspase-1 inhibitor Ac-YVAD-CMK inhibited the activation of caspase-1 and pyroptotic cell death. Ac-YVAD-CMK also reduced the lung injury, pulmonary edema and total protein in bronchoalveolar lavage fluid (BALF). In addition, Ac-YVAD-CMK significantly inhibited interleukin-&bgr; (IL-1&bgr;) release both in serum and BALF and reduced the levels of IL-18, tumor necrosis factor-&agr; (TNF-&agr;), High Mobility Group Box 1 (HMGB1) in BALF during LPS-induced ALI/ARDS. Conclusions:This study reported AM pyroptosis during LPS-induced ALI/ARDS in mice and has demonstrated that Ac-YVAD-CMK can prevent AM-induced pyroptosis and lung injury. These preliminary findings may form the basis for further studies to evaluate this pathway as a target for prevention or reduction of ALI/ARDS.

Intra-Peritoneal Administration of Mitochondrial DNA Provokes Acute Lung Injury and Systemic Inflammation via Toll-Like Receptor 9

The pathogenesis of sepsis is complex. Mitochondrial dysfunction, which is responsible for energy metabolism, intrinsic apoptotic pathway, oxidative stress, and systemic inflammatory responses, is closely related with severe sepsis induced death. Mitochondria DNA (mtDNA) contain un-methylated cytosine phosphate guanine (CpG) motifs, which exhibit immune stimulatory capacities. The aim of this study was to investigate the role and mechanism of mtDNA release on lipopolysaccharide (LPS) induced acute lung injury (ALI) and systemic inflammation. Following LPS injection, plasma mtDNA copies peak at 8 h. Compared with wild-type (WT) mice, mtDNA in toll like receptor 4 knockout (TLR4 KO) mice were significantly decreased. MtDNA intra-peritoneal administration causes apparent ALI as demonstrated by increased lung injury score, bronchoalveolar lavage fluid (BALF) total protein and wet/dry (W/D) ratio; mtDNA injection also directly provokes systemic inflammation, as demonstrated by increased IL-1β, IL-6, high-mobility group protein B1 (HMGB1) level; while nuclear DNA (nDNA) could not induce apparent ALI and systemic inflammation. However, compared with WT mice, TLR4 KO could not protect from mtDNA induced ALI and systemic inflammation. Specific TLR9 inhibitor, ODN 2088 pretreatment can significantly attenuate mtDNA induced ALI and systemic inflammation, as demonstrated by improved lung injury score, decreased lung wet/dry ratio, BALF total protein concentration, and decreased systemic level of IL-1β, IL-6 and HMGB1. MtDNA administration activates the expression of p-P38 mitogen-activated protein kinases (MAPK) in lung tissue and specific TLR9 inhibitor pretreatment can attenuate this activation. Thus, LPS-induced mtDNA release occurs in a TLR4-dependent manner, and mtDNA causes acute lung injury and systemic inflammation in a TLR9-dependent and TLR4-independent manner.

Nerve growth factor promotes expression of costimulatory molecules and release of cytokines in dendritic cells involved in Th2 response through LPS-induced p75NTR

Abstract Introduction: Nerve growth factor (NGF) plays an important role in asthmatic inflammatory responses. However, the effects of NGF on dendritic cells (DCs) in asthmatic inflammation remain unknown. Therefore, we examined the effects of NGF on co-stimulatory molecules and the release of cytokines after ovalbumin (OVA) and a low dose of LPS (low LPS) stimulation of dendritic cells. Methods: Bone-marrow-derived dendritic cells (BMDCs) were collected from 6- to 8-week-old wide or TLR4−/− mice. BMDCs were treated with OVA and/or low LPS for 12h, and then stimulated with NGF for 24h. ELISA and flow cytometry were performed to measure TSLP, IL-6, IL-10, and IL-12 production and MHCII and CD86 expression on BMDCs. BMDCs were exposed to p75 neurotrophin receptor (p75NTR) inhibitor (TAT-Pep5) or NF-kB inhibitor (QNZ) 30 min prior to NGF 1 h after NGF intervention, the levels of RelA and RelB in cytoplasmic and nuclear were detected by west blot. Co-cultured BMDCs with naïve CD4+ T cells, and ELISA was used to detect IL-4 and INF-γ levels. Results: NGF was found to markedly promote OVA and low LPS-induced expression of MHCII, CD86, secretion of TSLP and IL-6, and Th2-response-stimulating capacity of BMDCs. NGF affected BMDCs through LPS-induced p75NTR expression. TAT-Pep5 or QNZ could attenuate the promotive effect of NGF. Conclusions: NGF facilitates OVA with lowLPS-induced maturation of mouse BMDCs through LPS-up-regulated p75 NTR via activation of NF-κB pathways, providing another mechanism for the involvement of NGF in the Th2 response.

Role of interferon regulatory factor-1 in lipopolysaccharide-induced mitochondrial damage and oxidative stress responses in macrophages

Sepsis causes many early deaths; both macrophage mitochondrial damage and oxidative stress responses are key factors in its pathogenesis. Although the exact mechanisms responsible for sepsis-induced mitochondrial damage are unknown, the nuclear transcription factor, interferon regulatory factor-1 (IRF-1) has been reported to cause mitochondrial damage in several diseases. Previously, we reported that in addition to promoting systemic inflammation, IRF-1 promoted the apoptosis of and inhibited autophagy in macrophages. In the present study, we hypothesized that lipopolysaccharide (LPS)-induced IRF-1 activation in macrophages may promote mitochondrial damage and oxidative stress. In vitro, LPS was found to promote IRF-1 activation, reactive oxygen species (ROS) production, adenosine triphosphate (ATP) depletion, superoxide dismutase (SOD) consumption, malondialdehyde (MDA) accumulation and mitochondrial depolarization in macrophages in a time- and dose-dependent manner. These effects were abrogated in cells in which IRF-1 was knocked down. Furthermore, IRF-1 overexpression increased LPS-induced oxidative stress responses and mitochondrial damage. In vivo, peritoneal macrophages obtained from IRF-1 knockout (KO) mice produced less ROS and had less mitochondrial depolarization and damage following the administration of LPS, when compared to their wild-type (WT) counterparts. In addition, IRF-1 KO mice exhibited a decreased release of mitochondrial DNA (mtDNA) following the administration of LPS. Thus, IRF-1 may be a critical factor in augmenting LPS-induced oxidative stress and mitochondrial damage in macrophages.

Neutrophil extracellular traps contribute to the pathogenesis of acid-aspiration-induced ALI/ARDS

Background Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) is a manifestation of systemic inflammation in the lungs, but the factors that trigger inflammation in ALI/ARDS are unclear. We hypothesized that neutrophil extracellular traps (NETs) contribute to the pathogenesis of acid aspiration-induced ALI/ARDS. Results Analysis of bronchial aspirates from ARDS patients showed that NETs were significantly correlated with the degree of ARDS (r = –0.5846, p = 0.0359). NETs in bronchoalveolar lavage fluid of acid-aspiration mice were significantly higher (141.6 ± 23.08) at 3 h after injury than those in the sham group (1234 ± 101.9; p = 0.003, n = 5 per group). Exogenous NETs aggravated lung injury, while alvelestat and DNase markedly attenuated the intensity of ARDS. Materials and Methods We investigated whether NETs are involved in the severity of gastric aspiration-induced ARDS. Then, a hydrochloric acid aspiration-induced ALI murine model was used to assess whether NETs are pathogenic and whether targeting NETs is protective. Exogenous NETs were administered to mice. Alvelestat can inhibit neutrophil elastase (NE), which serves an important role in NET formation, so we investigated whether alvelestat could protect against ALI in cell and mouse models. Conclusions NETs may contribute to ALI/ARDS by promoting tissue damage and systemic inflammation. Targeting NETs by alvelestat may be a potential therapeutic strategy.

Neutrophil Extracellular Traps Are Pathogenic in Ventilator-Induced Lung Injury and Partially Dependent on TLR4

The pathogenesis of ventilator-induced lung injury (VILI) is associated with neutrophils. Neutrophils release neutrophil extracellular traps (NETs), which are composed of DNA and granular proteins. However, the role of NETs in VILI remains incompletely understood. Normal saline and deoxyribonuclease (DNase) were used to study the role of NETs in VILI. To further determine the role of Toll-like receptor 4 (TLR4) in NETosis, we evaluated the lung injury and NET formation in TLR4 knockout mice and wild-type mice that were mechanically ventilated. Some measures of lung injury and the NETs markers were significantly increased in the VILI group. DNase treatment markedly reduced NETs markers and lung injury. After high-tidal mechanical ventilation, the NETs markers in the TLR4 KO mice were significantly lower than in the WT mice. These data suggest that NETs are generated in VILI and pathogenic in a mouse model of VILI, and their formation is partially dependent on TLR4.

Pirfenidone ameliorates lipopolysaccharide-induced pulmonary inflammation and fibrosis by blocking NLRP3 inflammasome activation

HIGHLIGHTSFibroproliferation is present early in LPS‐induced acute lung injury.Pirfenidone ameliorates LPS‐induced pulmonary inflammation and fibrosis.The efficacy of pirfenidone is associated with inhibition of NLRP3 inflammasome activation. ABSTRACT Acute respiratory distress syndrome(ARDS)is a severe clinical disorder characterized by its acute onset, diffuse alveolar damage, intractable hypoxemia, and non‐cardiogenic pulmonary edema. Acute lung injury(ALI) can trigger persistent lung inflammation and fibrosis through activation of the NLRP3 inflammasome and subsequent secretion of mature IL‐1&bgr;, suggesting that the NLRP3 inflammasome is a potential therapeutic target for ALI, for which new therapeutic approaches are needed. Our present study aims to assess whether pirfenidone,with anti‐fibrotic and anti‐inflammatory properties, can improve LPS‐induced inflammation and fibrosis by inhibiting NLRP3 inflammasome activation. Male C57BL/6J mice were intratracheally injected with LPS to induce ALI. Mice were administered pirfenidone by oral gavage throughout the entire experimental course. The mouse macrophage cell line (J774A.1) was incubated with LPS and ATP, with or without PFD pre‐treatment. We demonstrated that PFD remarkably ameliorated LPS‐induced pulmonary inflammation and fibrosis and reduced IL‐1&bgr; and TGF‐&bgr;1 levels in bronchoalveolar lavage fluid(BALF). Pirfenidone substantially reduced NLRP3 and ASC expression and inhibited caspase‐1 activation and IL‐1&bgr; maturation in lung tissues. In vitro, the experiments revealed that PFD significantly suppressed LPS/ATP‐induced production of reactive oxygen species (ROS) and decreased caspase‐1 activation and the level of IL‐1&bgr; in J774A.1 cells. Taken together, the administration of PFD reduced LPS‐induced lung inflammation and fibrosis by blocking NLRP3 inflammasome activation and subsequent IL‐1&bgr; secretion. These findings indicated that PFD can down‐regulate NLRP3 inflammasome activation and that it may offer a promising therapeutic approach for ARDS patients.

The antimicrobial cathelicidin peptide hlF(1-11) attenuates alveolar macrophage pyroptosis induced by Acinetobacter baumannii in vivo

ABSTRACT Acinetobacter baumannii is a Gram‐negative coccobacillus found primarily in hospital settings that has recently emerged as a source of hospital‐acquired infections, including bacterial pneumonia. The hLF(1‐11) peptide comprising the first 11 N‐terminal residues of human lactoferrin exerts antimicrobial activity in vivo and was highly effective against multidrug‐resistant A. baumannii strains in vitro and in vivo. Pyroptosis is a caspase‐1‐dependent inflammatory cell death process and is induced by various microbial infections. In the present study, we investigated the molecular mechanisms that regulate pyroptosis induced by A. baumannii in macrophages. Our results revealed that A. baumannii induced pyroptosis through caspase‐1 activation and IL‐1&bgr; production. We also found that caspase‐1 activation and IL‐1&bgr; maturation in A. baumannii‐triggered pyroptotic cell death were reduced by hLF(1‐11) treatment. Moreover, hLF(1‐11) inhibited the A. baumannii‐induced caspase‐1 activation and pyroptosis of pulmonary alveolar macrophages in vivo.