Showing papers by "Jie Fan published in 2020"

Platelet-derived exosomes promote neutrophil extracellular trap formation during septic shock

Abstract: Platelets have been demonstrated to be potent activators of neutrophil extracellular trap (NET) formation during sepsis However, the mediators and molecular pathways involved in human platelet-mediated NET generation remain poorly defined Circulating plasma exosomes mostly originating from platelets may induce vascular apoptosis and myocardial dysfunction during sepsis; however, their role in NET formation remains unclear This study aimed to detect whether platelet-derived exosomes could promote NET formation during septic shock and determine the potential mechanisms involved Polymorphonuclear neutrophils (PMNs) were cocultured with exosomes isolated from the plasma of healthy controls and septic shock patients or the supernatant of human platelets stimulated ex vivo with phosphate buffer saline (PBS) or lipopolysaccharide (LPS) A lethal cecal ligation and puncture (CLP) mouse model was used to mimic sepsis in vivo; then, NET formation and molecular pathways were detected NET components (dsDNA and MPO-DNA complexes) were significantly increased in response to treatment with septic shock patient-derived exosomes and correlated positively with disease severity and outcome In the animal CLP model, platelet depletion reduced plasma exosome concentration, NET formation, and lung injury Mechanistic studies demonstrated that exosomal high-mobility group protein 1 (HMGB1) and/or miR-15b-5p and miR-378a-3p induced NET formation through the Akt/mTOR autophagy pathway Furthermore, the results suggested that IκB kinase (IKK) controls platelet-derived exosome secretion in septic shock Platelet-derived exosomes promote excessive NET formation in sepsis and subsequent organ injury This finding suggests a previously unidentified role of platelet-derived exosomes in sepsis and may lead to new therapeutic approaches

RAGE-induced ILC2 expansion in acute lung injury due to haemorrhagic shock

Abstract: Background Type 2 immune dysfunction contributes to acute lung injury and lethality following haemorrhagic shock (HS) and trauma. Group 2 innate lymphoid cells (ILC2s) play a significant role in the regulation of type 2 immune responses. However, the role of ILC2 in post-HS acute lung injury and the underlying mechanism has not yet been elucidated. Objective To investigate the regulatory role of ILC2s in HS-induced acute lung injury and the underlying mechanism in patients and animal model. Methods Circulating markers of type 2 immune responses in patients with HS and healthy controls were characterised. Using a murine model of HS, the role of high-mobility group box 1 (HMGB1)-receptor for advanced glycation end products (RAGE) signalling in regulation of ILC2 proliferation, survival and function was determined. And the role of ILC2 in inducing type 2 immune dysfunction was assessed as well. Results The number of ILC2s was significantly increased in the circulation of patients with HS that was correlated with the increase in the markers of type 2 immune responses in the patients. Animal studies showed that HMGB1 acted via RAGE to induce ILC2 accumulation in the lungs by promoting ILC2 proliferation and decreasing ILC2 death. The expansion of ILC2s resulted in type 2 cytokines secretion and eosinophil infiltration in the lungs, both of which contributed to lung injury after HS. Conclusions These results indicate that HMGB1-RAGE signalling plays a critical role in regulating ILC2 biological function that aggravates type 2 lung inflammation following HS.

Polymyxin for the treatment of intracranial infections of extensively drug-resistant bacteria in children after neurosurgical operation.

Abstract: Increased meningitis caused by extensively drug-resistant bacillary presents a significant challenge in antibiotic selection. The aim of our study was to evaluate the efficacy and safety of polymyxin in the treatment of post-neurosurgical meningitis due to the extensively drug-resistant bacillary in children. We performed a retrospective study on post-neurosurgical meningitis caused by the extensively drug-resistant bacillary in children, who were treated with polymyxin for ≥ 3 days. Among five post-neurosurgical meningitis cases that were included, the children were infected by Acinetobacter baumannii (n = 3), Klebsiella pneumonia (n = 1), and Pseudomonas aeruginosa (n = 1). The drug susceptibility test showed that they were extensively drug-resistant bacillary. Two patients received intravenous polymyxin E. Three children received intravenous combined with intraventricular injection of polymyxin B. One patient infected by Klebsiella pneumonia eventually died of septic shock. No serious adverse effects of polymyxin were observed. Polymyxin is a safe and effective therapy for post-neurosurgical, multidrug-resistant bacillary meningitis in children.

EGFR signaling augments TLR4 cell surface expression and function in macrophages via regulation of Rab5a activation.

Abstract: Toll-like receptor 4 (TLR4) is a key receptor sensing bacterial lipopolysaccharide (LPS), and is the most investigated member of the Toll-like receptor family (Kawai and Akira, 2007; Kayagaki et al., 2013; Klein et al., 2015). Cell surface TLR4 expression is determined by the balance between receptor trafficking from the Golgi apparatus to the cell membrane, and internalization of the cell surface receptor into endosomal compartments (Saltoh, 2009). In bone marrow-derived macrophages (BMDM), we observed LPS-induced EGFR phosphorylation on the surface of BMDM, and this was inhibited by pretreatment with PD168393 or TAPI-1 (Fig. S1). Next, we measured dynamic changes in cell surface TLR4 expression after LPS treatment. At 6, 12, and 24 h after LPS treatment, TLR4 expression on the surface of BMDM was increased ∼2-, ∼6-, and ∼9-fold, respectively, as compared with controls. EGFR inhibitor PD168393, however, inhibited LPS-mediated increases in cell surface expression of TLR4 at all time points (Fig. 1A and 1B). These alterations were also confirmed in a mouse macrophage cell line, RAW264.7 cells (Fig. S2). Then, C57BL/6 mice were injected intraperitoneally (i.p.) with LPS (10 mg/kg) with or without pretreatment of EGFR inhibitor, erlotinib (100 mg/kg B.W., gavage administration) at 30 min prior to LPS. At 24 h after LPS treatment, peritoneal macrophages were collected. LPS induced 5-fold increases in TLR4 expression on the macrophage surface, and erlotinib pretreatment inhibited TLR4 cell surface expression in response to LPS (Fig. 1C and 1D). Then, BMDM cells from EGFR mice were treated with LPS for 24 h and LPS treatment failed to induce increased cell surface expression of TLR4 in EGFR BMDM compared with WT BMDM cells (Fig. 1E and 1F). Similar findings were shown in vivo (Fig. 1G and 1H), where in contrast to WT mice, TLR4 surface expression on peritoneal macrophages from EGFR mice was not increased at 24 h after LPS challenge. These findings indicate that EGFR phosphorylation is essential for LPS-induced upregulation of TLR4 cell surface expression in macrophages. EGFR phosphorylation inhibitor, PD168393, effectively suppressed LPS-induced TLR4 phosphorylation (Fig. 1I). In addition, LPS-induced TLR4 phosphorylation was dramatically decreased in EGFR BMDM (Fig. 1J). We mutated TLR4 674 and 688 Tyr phosphorylation site into Ala. Then HEK293 cells were transfected with MD2, CD14, EGFR and TLR4 or TLR4 mutant. LPS treatment could not lead to the phosphorylation of EGFR in TLR4 mutant group (Fig. 1K and 1L). We also measured the effect of TLR4 phosphorylation on TLR4 cell membrane expression. 24 h after LPS treatment LPS-induced cell surface expression of TLR4 was markedly decreased in TLR4 mutant-expressing cells compared with cells expressing WT-TLR4 (Fig. 1M and 1N). LPS also induced co-localization of TLR4 and EGFR in BMDM at 30min after LPS treatment, and this was suppressed by EGFR phosphorylation inhibitor PD168393 (Fig. 1O). In addition, this kind of co-localization between TLR4 and EGFR in response to LPS also depended on the phosphorylation of TLR4 (Fig. S3). However, EGFR did not co-immunoprecipitate with TLR4 in control, LPS, or LPS plus PD168393 pretreatment (Fig. 1P). We further found that LPS significantly increased EGFR, but not TLR4, mRNA and total protein expression at 6, 12 and 24 h, and this was inhibited by PD168393 pretreatment (Fig. S4A–D). A large proportion of TLR4 receptors are stored in subcellular compartments, such as the Golgi apparatus and endosomes (Husebye et al., 2006). Since EGFR inhibitor decreased cell surface but not total TLR4 expression in response to LPS, we hypothesized that EGFR phosphorylation contributes to the transportation of TLR4 from the Golgi apparatus to the cell surface. Golgi marker GM130 applied to visualize the spatial relationship between the Golgi apparatus and TLR4 in BMDM. As shown in Fig. 1Q, LPS treatment reduced the colocalization of GM130 and TLR4, and PD168393 pretreatment partially restored the co-localization between Golgi and TLR4. Meanwhile, LPS lost its ability to reduce the co-localization between GM130 and TLR4 in EGFR BMDM (Fig. 1R). These data suggested that TLR4 is transported from Golgi to cell surface following LPS treatment and this is regulated by EGFR phosphorylation. Rab5a is an important downstream signaling molecule of EGFR and plays a critical role in actin remodeling, TLR4MyD88 interaction, and receptor internalization (Chen et al.,

Hepatic Estrogen Sulfotransferase Distantly Sensitizes Mice to Hemorrhagic Shock-Induced Acute Lung Injury.

Abstract: Hemorrhagic shock (HS) is a potential life-threatening condition that may lead to injury to multiple organs, including the lung. The estrogen sulfotransferase (EST, or SULT1E1) is a conjugating enzyme that sulfonates and deactivates estrogens. In this report, we showed that the expression of Est was markedly induced in the liver but not in the lung of female mice subject to HS and resuscitation. Genetic ablation or pharmacological inhibition of Est effectively protected female mice from HS-induced acute lung injury (ALI), including interstitial edema, neutrophil mobilization and infiltration, and inflammation. The pulmonoprotective effect of Est ablation or inhibition was sex-specific, because the HS-induced ALI was not affected in male Est-/- mice. Mechanistically, the pulmonoprotective phenotype in female Est-/- mice was accompanied by increased lung and circulating levels of estrogens, attenuated pulmonary inflammation, and inhibition of neutrophil mobilization from the bone marrow and neutrophil infiltration to the lung, whereas the pulmonoprotective effect was abolished upon ovariectomy, suggesting that the protection was estrogen dependent. The pulmonoprotective effect of Est ablation was also tissue specific, as loss of Est had little effect on HS-induced liver injury. Moreover, transgenic reconstitution of human EST in the liver of global Est-/- mice abolished the pulmonoprotective effect, suggesting that it is the EST in the liver that sensitizes mice to HS-induced ALI. Taken together, our results revealed a sex- and tissue-specific role of EST in HS-induced ALI. Pharmacological inhibition of EST may represent an effective approach to manage HS-induced ALI.

Correction to: EGFR signaling augments TLR4 cell surface expression and function in macrophages via regulation of Rab5a activation

Abstract: In the original publication the bands in Fig. 1J and Fig. 2B were not visible. The correct versions of Fig. 1J and Fig. 2B are provided in this correction.