The role of mitochondria in innate immunity and inflammation

PhD ThesisDeactivation of blood monocytes during sepsis is associated with increased mortality and susceptibility to secondary infections. Septic monocytes may also have mitochondrial DNA (mtDNA) depletion and mitochondrial respiratory dysfunction. Two principal approaches explored the link between these phenomena in THP-1 cells, a human leukaemia cell line resembling monocytes, to test the hypothesis that mtDNA depletion is important in the pathophysiology of monocytic cell immune deactivation. Firstly, the consequences of immune deactivation for mitochondria was assessed using an endotoxin tolerance model in which repeated exposures to lipopolysaccharide (LPS) trigger diminishing inflammatory responses. In parallel with the induction of endotoxin tolerance, LPS treatment lead to increased mitochondrial respiration due to the activation of mitochondrial biogenesis. These results could not be confirmed in healthy volunteers following inhalation of LPS as this model failed to induce endotoxin tolerance in blood monocytes. Secondly, the effects of depleting mtDNA, by treatment with ethidium bromide or transfection with short-interfering RNA targeted against mitochondrial transcription factor A, on immunity were measured. THP-1 cells with mtDNA depletion displayed the key phenotypic feature of deactivated septic monocytes, a decreased LPS-induced release of the pro-inflammatory cytokine tumour necrosis factor-α. Furthermore, there were significant alterations in the nuclear transcriptome of mtDNA-depleted THP-1 cells, with a particular inhibition of key innate immune signalling pathways and a marked blunting of the transcriptomic response to LPS. These investigations confirm that there are complex but vital links between mitochondria and innate immunity. Compensatory responses following an inflammatory insult include the simultaneous induction of mitochondrial biogenesis and shift to an anti-inflammatory phenotype. Moreover, when sepsis disrupts mitochondrial homeostasis the negative effects of mtDNA depletion on innate immunity may exacerbate monocyte immune deactivation. Further investigations should focus on exploring the fundamental processes coupling mitochondria with immunity and confirming these findings in blood monocytes during sepsis.Wellcome Trust Translational Medicine and Therapeutics Fellowshi

Spain and the Spaniards, in 1843

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Are attitudinal and perceptual body image the same or different? Evidence from high-level adaptation

We used a high-level adaptation paradigm to distinguish between two hypotheses: i) perceptual and attitudinal body image measurements reflect conceptually different mechanisms which are statistically independent of each other; ii) attitudinal (e.g., questionnaire) and perceptual (e.g., visual yes-no) body image tasks represent two different ways of measuring exactly the same construct. Forty women, with no history of eating disorders, carried out the experiment. Each participant carried out five adaptation blocks, with adapting stimuli representing female bodies at: extreme-low body mass index (BMI), mid-low BMI, actual BMI of the observer, mid-high BMI and extreme-high BMI. Block order was randomized across participants. The main outcome variable was percentage error in participants’ self-estimates of body size, measured post-adaption. In regressions of this percentage error on the strength of the adapting stimuli together with observers’ attitudinal body image as a covariate, we found positive regression slopes and no evidence for any interaction between the fixed effects. Therefore, we conclude that perceptual and attitudinal body image mechanisms are indeed independent of each other. In the light of this evidence, we discuss how people with eating disorders, like anorexia nervosa, may come to over-estimate their body size

Exposure of Monocytic Cells to Lipopolysaccharide Induces Coordinated Endotoxin Tolerance, Mitochondrial Biogenesis, Mitophagy, and Antioxidant Defenses

In order to limit the adverse effects of excessive inflammation, anti-inflammatory responses are stimulated at an early stage of an infection, but during sepsis these can lead to deactivation of immune cells including monocytes. In addition, there is emerging evidence that the up-regulation of mitochondrial quality control mechanisms, including mitochondrial biogenesis and mitophagy, is important during the recovery from sepsis and inflammation. We aimed to describe the relationship between the compensatory immune and mitochondrial responses that are triggered following exposure to an inflammatory stimulus in human monocytic cells. Incubation with lipopolysaccharide resulted in a change in the immune phenotype of THP-1 cells consistent with the induction of endotoxin tolerance, similar to that seen in deactivated septic monocytes. After exposure to LPS there was also early evidence of oxidative stress, which resolved in association with the induction of antioxidant defenses and the stimulation of mitochondrial degradation through mitophagy. This was compensated by a parallel up-regulation of mitochondrial biogenesis that resulted in an overall increase in mitochondrial respiratory activity. These observations improve our understanding of the normal homeostatic responses that limit the adverse cellular effects of unregulated inflammation, and which may become ineffective when an infection causes sepsis

Modulation of inflammation by anti-TNF α mAb-dendrimer nanoparticles loaded in tyramine-modified gellan gum hydrogels in a cartilage-on-a-chip model

Rheumatoid arthritis (RA) is an autoimmune and chronic inflammatory disease characterized by joint inflammation. Since the inflammatory condition plays an important role in the disease process, it is important to develop and test new therapeutic approaches that specifically target and treat joint inflammation. In this study, a human 3D inflammatory cartilage-on-a-chip model was established to test the therapeutic efficacy of anti-TNFα mAb-CS/PAMAM dendrimer NPs loaded-Tyramine-Gellan Gum in the treatment of inflammation. The results showed that the proposed therapeutic approach applied to the human monocyte cell line (THP-1) and human chondrogenic primary cells (hCH) cell-based inflammation system revealed an anti-inflammatory capacity that increased over 14 days. It was also possible to observe that Coll type II was highly expressed by inflamed hCH upon the culture with anti-TNF α mAb-CS/PAMAM dendrimer NPs, indicating that the hCH cells were able maintain their biological function. The developed preclinical model allowed us to provide more robust data on the potential therapeutic effect of anti-TNF α mAb-CS/PAMAM dendrimer NPs loaded-Ty-GG hydrogel in a physiologically relevant model.The authors thank the financial support under the Norte2020 project (NORTE-08-5369-FSE000044). M. R. C. acknowledges TERM RES Hub Ref. Norte-01-0145-FEDER-02219015 working contract. D. C. F. acknowledges Portuguese Foundation for Science and Technology (FCT) for his phD scholarship (PD/ BD/143081/2018) and F. R. M. for her contract under the Transitional Rule DL 57/2016 (CTTI-57/18-I3BS(5)). C. M. A., D. C. V. and S. C. K. thank the support of FCT (PTDC/BTM-ORG/ 28070/2017). D. C. V acknowledges the CEEC individual contract (CEECIND/00352/2017). S. C. K wishes to record the financial support from EU Framework Programme for Research and Innovation H2020 on FoReCaST under grant agreement no. 668983 and BREAST-IT FCT-Portugal project (PTDC/BTM-ORG/ 28168/2017). The FCT distinction attributed to J. M. O. under the Investigator FCT program (number IF/01285/2015) is also greatly acknowledge

Mitochondrial DNA depletion induces innate immune dysfunction rescued by interferon-γ

Monocytes from sepsis patients have mitochondrial DNA (mtDNA) depletion and immune deactivation. Here we find that THP-1 cell immune responses are impaired by experimentally depleting mtDNA, through dysregulated immune signalling, and rescued by interferon-γ treatment

Randomised controlled trial of GM-CSF in critically ill patients with impaired neutrophil phagocytosis

Background. Critically ill patients with impaired neutrophil phagocytosis have significantly increased risk of nosocomial infection. Granulocyte-macrophage colony-stimulating factor (GM-CSF) improves phagocytosis by neutrophils ex vivo. This study tested the hypothesis that GM-CSF improves neutrophil phagocytosis in critically ill patients in whom phagocytosis is known to be impaired Methods. This was a multi-centre, phase 2a randomised, placebo-controlled clinical trial Using a personalised medicine approach, only critically ill patients with impaired neutrophil phagocytosis were included. Patients were randomised 1:1 to subcutaneous GM-CSF (3 microgrammws/kg/day) or placebo, once daily for 4 days. The primary outcome measure was neutrophil phagocytosis 2 days after initiation of GM-CSF. Secondary outcomes included neutrophil phagocytosis over time, neutrophil functions other than phagocytosis, monocyte HLA-DR expression, and safety. Results. Thirty-eight patients were recruited from 5 intensive care units (17 randomised to GM-CSF). Mean neutrophil phagocytosis at day 2 was 57.2% (SD 13.2%) in the GM-CSF group and 49.8% (13.4%) in the placebo group, p=0.73. The proportion of patients with neutrophil phagocytosis >50% at day 2, and monocyte HLA-DR, appeared significantly higher in the GM-CSF group. Neutrophil functions other than phagocytosis did not appear significantly different between the groups. The most common adverse event associated with GM-CSF was pyrexia. Conclusions. GM-CSF did not improve mean neutrophil phagocytosis at day 2, but was safe and appeared to increase the proportion of patients with adequate phagocytosis. The study suggests proof of principle for a pharmacological effect on neutrophil function in a subset of critically ill patients.This work was funded by a grant from the Medical Research Council (G1100233), with additional support from the National Institute for Health Research (NIHR) Newcastle Biomedical Research Centre. It was sponsored by Newcastle Universit

Diagnostic accuracy of pulmonary host inflammatory mediators in the exclusion of ventilator-acquired pneumonia.

BACKGROUND: Excessive use of empirical antibiotics is common in critically ill patients. Rapid biomarker-based exclusion of infection may improve antibiotic stewardship in ventilator-acquired pneumonia (VAP). However, successful validation of the usefulness of potential markers in this setting is exceptionally rare. OBJECTIVES: We sought to validate the capacity for specific host inflammatory mediators to exclude pneumonia in patients with suspected VAP. METHODS: A prospective, multicentre, validation study of patients with suspected VAP was conducted in 12 intensive care units. VAP was confirmed following bronchoscopy by culture of a potential pathogen in bronchoalveolar lavage fluid (BALF) at >10(4) colony forming units per millilitre (cfu/mL). Interleukin-1 beta (IL-1β), IL-8, matrix metalloproteinase-8 (MMP-8), MMP-9 and human neutrophil elastase (HNE) were quantified in BALF. Diagnostic utility was determined for biomarkers individually and in combination. RESULTS: Paired BALF culture and biomarker results were available for 150 patients. 53 patients (35%) had VAP and 97 (65%) patients formed the non-VAP group. All biomarkers were significantly higher in the VAP group (p<0.001). The area under the receiver operator characteristic curve for IL-1β was 0.81; IL-8, 0.74; MMP-8, 0.76; MMP-9, 0.79 and HNE, 0.78. A combination of IL-1β and IL-8, at the optimal cut-point, excluded VAP with a sensitivity of 100%, a specificity of 44.3% and a post-test probability of 0% (95% CI 0% to 9.2%). CONCLUSIONS: Low BALF IL-1β in combination with IL-8 confidently excludes VAP and could form a rapid biomarker-based rule-out test, with the potential to improve antibiotic stewardship

Adjunctive rifampicin for Staphylococcus aureus bacteraemia (ARREST): a multicentre, randomised, double-blind, placebo-controlled trial.

BACKGROUND: Staphylococcus aureus bacteraemia is a common cause of severe community-acquired and hospital-acquired infection worldwide. We tested the hypothesis that adjunctive rifampicin would reduce bacteriologically confirmed treatment failure or disease recurrence, or death, by enhancing early S aureus killing, sterilising infected foci and blood faster, and reducing risks of dissemination and metastatic infection. METHODS: In this multicentre, randomised, double-blind, placebo-controlled trial, adults (≥18 years) with S aureus bacteraemia who had received ≤96 h of active antibiotic therapy were recruited from 29 UK hospitals. Patients were randomly assigned (1:1) via a computer-generated sequential randomisation list to receive 2 weeks of adjunctive rifampicin (600 mg or 900 mg per day according to weight, oral or intravenous) versus identical placebo, together with standard antibiotic therapy. Randomisation was stratified by centre. Patients, investigators, and those caring for the patients were masked to group allocation. The primary outcome was time to bacteriologically confirmed treatment failure or disease recurrence, or death (all-cause), from randomisation to 12 weeks, adjudicated by an independent review committee masked to the treatment. Analysis was intention to treat. This trial was registered, number ISRCTN37666216, and is closed to new participants. FINDINGS: Between Dec 10, 2012, and Oct 25, 2016, 758 eligible participants were randomly assigned: 370 to rifampicin and 388 to placebo. 485 (64%) participants had community-acquired S aureus infections, and 132 (17%) had nosocomial S aureus infections. 47 (6%) had meticillin-resistant infections. 301 (40%) participants had an initial deep infection focus. Standard antibiotics were given for 29 (IQR 18-45) days; 619 (82%) participants received flucloxacillin. By week 12, 62 (17%) of participants who received rifampicin versus 71 (18%) who received placebo experienced treatment failure or disease recurrence, or died (absolute risk difference -1·4%, 95% CI -7·0 to 4·3; hazard ratio 0·96, 0·68-1·35, p=0·81). From randomisation to 12 weeks, no evidence of differences in serious (p=0·17) or grade 3-4 (p=0·36) adverse events were observed; however, 63 (17%) participants in the rifampicin group versus 39 (10%) in the placebo group had antibiotic or trial drug-modifying adverse events (p=0·004), and 24 (6%) versus six (2%) had drug interactions (p=0·0005). INTERPRETATION: Adjunctive rifampicin provided no overall benefit over standard antibiotic therapy in adults with S aureus bacteraemia. FUNDING: UK National Institute for Health Research Health Technology Assessment