Aaron Scott

SLPI and elafin: multifunctional antiproteases of the WFDC family

SLPI (secretory leucoprotease inhibitor) and elafin represent the archetypal members of the WFDC [WAP (whey acidic protein) four disulfide core] family of proteins, and were originally characterized as protease inhibitors but have since been shown to possess a wider repertoire of activities. These functions include antimicrobial and immunomodulatory properties, suggesting that these proteins may play key roles in the innate immune response, and indicate the potential to develop some of these proteins as novel therapeutics. Susceptibility to host and bacterial protease cleavage may, however, limit the efficacy of recombinant protein therapies in diseases with a high protease burden such as CF (cystic fibrosis) lung disease. To overcome this problem, further refinement of the native proteins will be required to provide effective treatment strategies.

Evaluation of the Ability of LL-37 to Neutralise LPS In Vitro and Ex Vivo

Background Human cathelicidin LL-37 is a cationic antimicrobial peptide (AMP) which possesses a variety of activities including the ability to neutralise endotoxin. In this study, we investigated the role of LPS neutralisation in mediating LL-37s ability to inhibit Pseudomonas aeruginosa LPS signalling in human monocytic cells. Methodology/Principal Findings Pre-treatment of monocytes with LL-37 significantly inhibited LPS-induced IL-8 production and the signalling pathway of associated transcription factors such as NF-κB. However, upon removal of LL-37 from the media prior to LPS stimulation, these inhibitory effects were abolished. These findings suggest that the ability of LL-37 to inhibit LPS signalling is largely dependent on extracellular LPS neutralisation. In addition, LL-37 potently inhibited cytokine production induced by LPS extracted from P. aeruginosa isolated from the lungs of cystic fibrosis (CF) patients. In the CF lung, polyanionic molecules such as glycosaminoglycans (GAGs) and DNA bind LL-37 and impact negatively on its antibacterial activity. In order to determine whether such interactions interfere with the LPS neutralising ability of LL-37, the status of LL-37 and its ability to bind LPS in CF sputum were investigated. Overall our findings suggest that in the CF lung, the ability of LL-37 to bind LPS and inhibit LPS-induced IL-8 production is attenuated as a result of binding to DNA and GAGs. However, LL-37 levels and its concomitant LPS-binding activity can be increased with a combination of DNase and GAG lyase (heparinase II) treatment. Conclusions/Significance Overall, these findings suggest that a deficiency in available LL-37 in the CF lung may contribute to greater LPS-induced inflammation during CF lung disease.

Altered Toll-like Receptor 2-mediated Endotoxin Tolerance Is Related to Diminished Interferon β Production

Induction of endotoxin tolerance leads to a reduced inflammatory response after repeated challenge by LPS and is important for resolution of inflammation and prevention of tissue damage. Enterobacterial LPS is recognized by the TLR4 signaling complex, whereas LPS of some non-enterobacterial organisms is capable of signaling independently of TLR4 utilizing TLR2-mediated signal transduction instead. In this study we report that Porphyromonas gingivalis LPS, a TLR2 agonist, fails to induce a fully endotoxin tolerant state in a human monocytic cell line (THP-1) and mouse bone marrow-derived macrophages. In contrast to significantly decreased production of human IL-8 and TNF-α and, in mice, keratinocyte-derived cytokine (KC), macrophage inflammatory protein-2 (MIP-2), and TNF-α after repeated challenge with Escherichia coli LPS, cells repeatedly exposed to P. gingivalis LPS responded by producing less TNF-α but sustained elevated secretion of IL-8, KC, and MIP-2. Furthermore, in endotoxin-tolerant cells, production of IL-8 is controlled at the signaling level and correlates well with NF-κB activation, whereas TNF-α expression is blocked at the gene transcription level. Interferon β plays an important role in attenuation of chemokine expression in endotoxin-tolerized cells as shown in interferon regulatory factor-3 knock-out mice. In addition, human gingival fibroblasts, commonly known not to display LPS tolerance, were found to be tolerant to repeated challenge by LPS if pretreated with interferon β. The data suggest that the inability of the LPS-TLR2 complex to induce full endotoxin tolerance in monocytes/macrophages is related to diminished production of interferon β and may partly explain the involvement of these LPS isoforms in the pathogenesis of chronic inflammatory diseases.

Vitamin D deficiency in human and murine sepsis

Objectives: Vitamin D deficiency has been implicated as a pathogenic factor in sepsis and ICU mortality but causality of these associations has not been demonstrated. To determine whether sepsis and severe sepsis are associated with vitamin D deficiency and to determine whether vitamin D deficiency influences the severity of sepsis. Design, Setting, and Patients: Sixty-one patients with sepsis and severe sepsis from two large U.K. hospitals and 20 healthy controls were recruited. Murine models of cecal ligation and puncture and intratracheal lipopolysaccharide were undertaken in normal and vitamin D deficient mice to address the issue of causality. Measurements and Main Results: Patients with severe sepsis had significantly lower concentrations of 25-hydroxyvitamin D3 than patients with either mild sepsis or age-matched healthy controls (15.7 vs 49.5 vs 66.5 nmol/L; p = 0.0001). 25-hydroxyvitamin D3 concentrations were significantly lower in patients who had positive microbiologic culture than those who were culture negative (p = 0.0023) as well as those who died within 30 days of hospital admission (p = 0.025). Vitamin D deficiency in murine sepsis was associated with increased peritoneal (p = 0.037), systemic (p = 0.019), and bronchoalveolar lavage (p = 0.011) quantitative bacterial culture. This was associated with reduced local expression of the cathelicidin-related antimicrobial peptide as well as evidence of defective macrophage phagocytosis (p = 0.029). In the intratracheal lipopolysaccharide model, 1,500 IU of intraperitoneal cholecalciferol treatment 6 hours postinjury reduced alveolar inflammation, cellular damage, and hypoxia. Conclusions: Vitamin D deficiency is common in severe sepsis. This appears to contribute to the development of the condition in clinically relevant murine models and approaches to correct vitamin D deficiency in patients with sepsis should be developed.

A role for whey acidic protein four-disulfide-core 12 (WFDC12) in the regulation of the inflammatory response in the lung

Introduction Secretory leucocyte protease inhibitor and elafin are members of the whey acidic protein (WAP), or WAP four disulfide-core (WFDC), family of proteins and have multiple contributions to innate defence including inhibition of neutrophil serine proteases and inhibition of the inflammatory response to lipopolysaccharide (LPS). This study aimed to explore potential activities of WFDC12, a previously uncharacterised WFDC protein expressed in the lung. Methods Recombinant expression and purification of WFDC12 were optimised in Escherichia coli. Antiprotease, antibacterial and immunomodulatory activities of recombinant WFDC12 were evaluated and levels of endogenous WFDC12 protein were characterised by immunostaining and ELISA. Results Recombinant WFDC12 inhibited cathepsin G, but not elastase or proteinase-3 activity. Monocytic cells pretreated with recombinant WFDC12 before LPS stimulation produced significantly lower levels of the pro-inflammatory cytokines interleukin-8 and monocyte chemotactic protein-1 compared with cells stimulated with LPS alone. Recombinant WFDC12 became conjugated to fibronectin in a transglutaminase-mediated reaction and retained antiprotease activity. In vivo WFDC12 expression was confirmed by immunostaining of human lung tissue sections. WFDC12 levels in human bronchoalveolar lavage fluid from healthy and lung-injured patients were quantitatively compared, showing WFDC12 to be elevated in both patients with acute respiratory distress syndrome and healthy subjects treated with LPS, relative to healthy controls. Conclusions Together, these results suggest a role for this lesser known WFDC protein in the regulation of lung inflammation.

Pro-inflammatory effects of e-cigarette vapour condensate on human alveolar macrophages

Objective Vaping may increase the cytotoxic effects of e-cigarette liquid (ECL). We compared the effect of unvaped ECL to e-cigarette vapour condensate (ECVC) on alveolar macrophage (AM) function. Methods AMs were treated with ECVC and nicotine-free ECVC (nfECVC). AM viability, apoptosis, necrosis, cytokine, chemokine and protease release, reactive oxygen species (ROS) release and bacterial phagocytosis were assessed. Results Macrophage culture with ECL or ECVC resulted in a dose-dependent reduction in cell viability. ECVC was cytotoxic at lower concentrations than ECL and resulted in increased apoptosis and necrosis. nfECVC resulted in less cytotoxicity and apoptosis. Exposure of AMs to a sub-lethal 0.5% ECVC/nfECVC increased ROS production approximately 50-fold and significantly inhibited phagocytosis. Pan and class one isoform phosphoinositide 3 kinase inhibitors partially inhibited the effects of ECVC/nfECVC on macrophage viability and apoptosis. Secretion of interleukin 6, tumour necrosis factor α, CXCL-8, monocyte chemoattractant protein 1 and matrix metalloproteinase 9 was significantly increased following ECVC challenge. Treatment with the anti-oxidant N-acetyl-cysteine (NAC) ameliorated the cytotoxic effects of ECVC/nfECVC to levels not significantly different from baseline and restored phagocytic function. Conclusions ECVC is significantly more toxic to AMs than non-vaped ECL. Excessive production of ROS, inflammatory cytokines and chemokines induced by e-cigarette vapour may induce an inflammatory state in AMs within the lung that is partly dependent on nicotine. Inhibition of phagocytosis also suggests users may suffer from impaired bacterial clearance. While further research is needed to fully understand the effects of e-cigarette exposure in humans in vivo, we caution against the widely held opinion that e-cigarettes are safe.

T2 Vitamin D supplementation reduces perioperative systemic and alveolar inflammation in patients undergoing oesophagectomy: Results of the Vindaloo Trial

Vitamin D deficiency is associated with increased risk of ARDS post-oesophagectomy. We recruited patients to a double-blind, randomised controlled trial of high dose Vitamin D supplementation 3–14 days pre-oesophagectomy. 79 patients were randomised to receive placebo or 300,000 IU oral Vitamin D liquid 3–14 days prior to oesophagectomy. Blood samples were collected pre-dose, post-dose (pre-op) and post-op and analysed for 25-OH and 1,25-dOH Vitamin D, inflammatory cells and cytokines. Broncho-alveolar lavage fluid was collected at the end of the operation. PICCO biomarkers of alveolar capillary damage (EVLWI and PVPI) were measured pre- and post-op. Pre-operative supplementation with Vitamin D was well tolerated with no SUSARs and significantly increased circulating 25-OH and 1,25-OH Vitamin D (p < 0.0001). This was associated with reduced systemic inflammation (IL-6 (p = 0.02) and IL-8 (p = 0.002)) and an increase in circulatory Treg (p = 0.027). Changes in PICCO biomarkers were lower in supplemented patients suggesting lower perioperative alveolar oedema (EVLWI p = 0.05, PVPI p = 0.04). This did not result in a significant difference in oxygenation at 24 h. Post-op, systemic and alveolar alarmin (IL-1B, IL-6, IL-8) response was similar in treated and untreated patients but the systemic release of IL-1ra (p = 0.046), sTNFR-1 (p = 0.05) and s-TNFR-2 (p = 0.02) were elevated in treated patients. There was also evidence of decreased alveolar macrophage efferocytosis in patients with Vitamin D deficiency (p = 0.003). Clinical diagnoses of ARDS were significantly lower in this cohort than in previous cohorts, but the study was not powered to detect that outcome. Mortality post-operative was not significantly different at 30 or 90 days but there is a significant difference after 300 days of follow-up (placebo 33% mortality, Vitamin D 8% mortality p = 0.033). In conclusion, vitamin D supplementation was a safe, well tolerated pre-operative intervention that reduced systemic inflammation and biomarkers of alveolar oedema. With evidence of enhanced anti-inflammatory mechanism that may have influenced longer term post-operative survival, Vitamin D deficiency should be identified and treated in patients at risk of ARDS.

Sepsis Induces a Dysregulated Neutrophil Phenotype That Is Associated with Increased Mortality

Background Neutrophil dysfunction in sepsis has been implicated in the pathogenesis of multiorgan failure; however, the role of neutrophil extracellular traps (NETs) remains uncertain. We aimed to determine the sequential changes in ex vivo NETosis and its relationship with mortality in patients with sepsis and severe sepsis. Methods This was a prospective observational cohort study enrolling 21 healthy age-matched controls and 39 sepsis and 60 severe sepsis patients from acute admissions to two UK hospitals. Patients had sequential bloods for the ex vivo assessment of NETosis in response to phorbol-myristate acetate (PMA) using a fluorometric technique and chemotaxis using time-lapse video microscopy. Continuous data was tested for normality, with appropriate parametric and nonparametric tests, whilst categorical data was analysed using a chi-squared test. Correlations were performed using Spearmans rho. Results Ex vivo NETosis was reduced in patients with severe sepsis, compared to patients with sepsis and controls (p = 0.002). PMA NETosis from patients with septic shock was reduced further (p < 0.001) compared to controls. The degree of metabolic acidosis correlated with reduced NETosis (p < 0.001), and this was replicated when neutrophils from healthy donors were incubated in acidotic media. Reduced NETosis at baseline was associated with an increased 30-day (p = 0.002) and 90-day mortality (p = 0.014) in sepsis patients. These findings were accompanied by defects in neutrophil migration and delayed apoptosis. Resolution of sepsis was not associated with the return to baseline levels of NETosis or migration. Conclusions Sepsis induces significant changes in neutrophil function with the degree of dysfunction corresponding to the severity of the septic insult which persists beyond physiological recovery from sepsis. The changes induced lead to the failure to effectively contain and eliminate the invading pathogens and contribute to sepsis-induced immunosuppression. For the first time, we demonstrate that reduced ex vivo NETosis is associated with poorer outcomes from sepsis.

LSC - 2017 - Effects of e-cigarette vapour condensate upon human alveolar macrophage function

The process of vaping may increase the cytotoxic effects of e-cigarette liquid (ECL) and increase the negative effects on alveolar macrophages yet many studies have used only unvaped fluid. We sought to examine the potential cytotoxic and functional effects of vaped and unvaped e-cigarette fluid on alveolar macrophages (AM). AMs were treated with e-cigarette vapour condensate (ECVC)/ECL +/- nicotine. Viability, apoptosis/necrosis, cytokine release, phagocytosis and ROS generation were then assessed. AM culture with ECL/ECVC resulted in dose dependent reduction in cell viability. ECVC was cytotoxic at lower concentrations than ECL (0.8% vs 5%, n=6). 24hr culture with 0.8% ECVC resulted in a 25% increase in apoptosis and necrosis (p ECVC is significantly more toxic to AMs than non-vaped ECL. Elevated inflammatory cytokine production and ROS suggests ECIG users may be inducing a chronic inflammatory state in vapour exposed AMs. Impaired phagocytic ability following ECVC exposure suggests AMs in the lung of ECIG users may show impaired bacterial clearance. Taken together this suggests ECIG users may experience more frequent and more severe bacterial infections due to impaired innate immune response by AMs.

Characterisation of eppin function: expression and activity in the lung

Eppin is a serine protease inhibitor expressed in male reproductive tissues. The aim of this study was to investigate the localisation and regulation of eppin expression in myeloid and epithelial cell lines, and explore its potential role as a multifunctional host defence protein. Using immunohistochemistry and Western blotting, eppin was detected in the lungs of patients with acute respiratory distress syndrome and cystic fibrosis lung disease. Expression of eppin in monocytic cells was unaffected by stimulation with Toll-like receptor agonists, cytokines and hormone receptor agonists. However, upregulated expression and secretion of eppin was observed following treatment of monocytes with epidermal growth factor. Incubation of recombinant eppin with monocytic cells resulted in significant inhibition of lipopolysaccharide-induced chemokine production. Furthermore, eppin inhibited lipopolysaccharide-induced NF-κB activation by a mechanism which involved accumulation of phosphorylated IκBα. In an in vivo model of lung inflammation induced by lipopolysaccharide, eppin administration resulted in decreased recruitment of neutrophils to the lung with a concomitant reduction in the levels of the neutrophil chemokine macrophage inflammatory protein-2. Overall, these results suggest a role for eppin outside of the reproductive tract and that eppin may have a role in the innate immune response in the lung. Eppin is a low-molecular-weight protein which is expressed in the human lung during inflammation http://ow.ly/WZuQ30aELEI