Macrophage Subset Sensitivity to Endotoxin Tolerisation by Porphyromonas gingivalis

Macrophages (MΦs) determine oral mucosal responses; mediating tolerance to commensal microbes and food whilst maintaining the capacity to activate immune defences to pathogens. MΦ responses are determined by both differentiation and activation stimuli, giving rise to two distinct subsets; pro-inflammatory M1- and anti-inflammatory/regulatory M2- MΦs. M2-like subsets predominate tolerance induction whereas M1 MΦs predominate in inflammatory pathologies, mediating destructive inflammatory mechanisms, such as those in chronic P.gingivalis (PG) periodontal infection. MΦ responses can be suppressed to benefit either the host or the pathogen. Chronic stimulation by bacterial pathogen associated molecular patterns (PAMPs), such as LPS, is well established to induce tolerance. The aim of this study was to investigate the susceptibility of MΦ subsets to suppression by P. gingivalis. CD14hi and CD14lo M1- and M2-like MΦs were generated in vitro from the THP-1 monocyte cell line by differentiation with PMA and vitamin D3, respectively. MΦ subsets were pre-treated with heat-killed PG (HKPG) and PG-LPS prior to stimulation by bacterial PAMPs. Modulation of inflammation was measured by TNFα, IL-1β, IL-6, IL-10 ELISA and NFκB activation by reporter gene assay. HKPG and PG-LPS differentially suppress PAMP-induced TNFα, IL-6 and IL-10 but fail to suppress IL-1β expression in M1 and M2 MΦs. In addition, P.gingivalis suppressed NFκB activation in CD14lo and CD14hi M2 regulatory MΦs and CD14lo M1 MΦs whereas CD14hi M1 pro-inflammatory MΦs were refractory to suppression. In conclusion, P.gingivalis selectively tolerises regulatory M2 MΦs with little effect on pro-inflammatory CD14hi M1 MΦs; differential suppression facilitating immunopathology at the expense of immunity

Erratum to: Methods for evaluating medical tests and biomarkers

[This corrects the article DOI: 10.1186/s41512-016-0001-y.]

Erratum to: Methods for evaluating medical tests and biomarkers

[This corrects the article DOI: 10.1186/s41512-016-0001-y.]

Evidence synthesis to inform model-based cost-effectiveness evaluations of diagnostic tests: a methodological systematic review of health technology assessments

Background: Evaluations of diagnostic tests are challenging because of the indirect nature of their impact on patient outcomes. Model-based health economic evaluations of tests allow different types of evidence from various sources to be incorporated and enable cost-effectiveness estimates to be made beyond the duration of available study data. To parameterize a health-economic model fully, all the ways a test impacts on patient health must be quantified, including but not limited to diagnostic test accuracy. Methods: We assessed all UK NIHR HTA reports published May 2009-July 2015. Reports were included if they evaluated a diagnostic test, included a model-based health economic evaluation and included a systematic review and meta-analysis of test accuracy. From each eligible report we extracted information on the following topics: 1) what evidence aside from test accuracy was searched for and synthesised, 2) which methods were used to synthesise test accuracy evidence and how did the results inform the economic model, 3) how/whether threshold effects were explored, 4) how the potential dependency between multiple tests in a pathway was accounted for, and 5) for evaluations of tests targeted at the primary care setting, how evidence from differing healthcare settings was incorporated. Results: The bivariate or HSROC model was implemented in 20/22 reports that met all inclusion criteria. Test accuracy data for health economic modelling was obtained from meta-analyses completely in four reports, partially in fourteen reports and not at all in four reports. Only 2/7 reports that used a quantitative test gave clear threshold recommendations. All 22 reports explored the effect of uncertainty in accuracy parameters but most of those that used multiple tests did not allow for dependence between test results. 7/22 tests were potentially suitable for primary care but the majority found limited evidence on test accuracy in primary care settings. Conclusions: The uptake of appropriate meta-analysis methods for synthesising evidence on diagnostic test accuracy in UK NIHR HTAs has improved in recent years. Future research should focus on other evidence requirements for cost-effectiveness assessment, threshold effects for quantitative tests and the impact of multiple diagnostic tests

Peptidoglycan differentially cross-tolerises <i>P. gingivalis</i>-stimulated CD14^hi/lo M1 and M2 MΦ subsets.

<p>CD14^hi/lo M1 and M2 MΦ subsets exhibit a differential cross-tolerisation of cytokine production and NFκB activity to the <i>P gingivalis</i> PAMPs, PG-LPS and HKPG. CD14^lo M1, CD14^hi M1, CD14^lo M2 and CD14^hi M2 MΦ subsets were pre-stimulated (tolerised) with 10µg/ml peptidoglycan (PGN) for 24 hours prior to stimulation with PG-LPS or HKPG and incubated for a further 18 hours. Tolerisation/suppression of the pro-inflammatory cytokines (TNFα, IL-1β and IL-6), the anti-inflammatory cytokine (IL-10) and the transcription factor activity (NFκB) is expressed as the mean percentage suppression ± SD of non-tolerised stimulation controls. Data displayed represents triplicate samples for n = 3 replicate experiments.</p

<i>P. gingivalis</i> differentially suppresses CD14^hi/lo M1 and M2 MΦ NFκB activity.

<p>CD14^lo M1 (a) CD14^hi M1 (b), CD14^lo M2 (c) and CD14^hi M2 (d) MΦ subsets were pre-stimulated (tolerised) with either 100 ng/ml PG-LPS (unshaded) or 1×10⁷ cells/ml HKPG (shaded) for 24 hours prior to stimulation with PG-LPS or HKPG and incubated for a further 18 hours (untolerised controls indicated in bold). NFκB activation is expressed as the mean absorbance units A_620nm ± SD for the CD14^hi/lo M1 and M2 MΦ subsets. Data displayed represents triplicate samples for n = 3 replicate experiments. Significant effects compared to the un-tolerised stimulus control (bold) for each MΦ subset are indicated as *p<0.05, ***p<0.001 and ns, not significant.</p

M1 & M2 MΦs display differential cytokine profiles in response to PG-LPS and HKPG.

<p>THP-1-derived M1 and M2 MΦs were generated by differentiating THP-1 monocytes with either 25 ng/ml phorbol 12-myristate 13-acetate (PMA) for 3 days or 10 nM 1,25-(OH)₂ vitamin D₃ for 7 days, respectively. M1 (bold) and M2 (shaded) MΦ subsets were stimulated with either 100 ng/ml PG-LPS (a, b and c) or 1×10⁷ cells/ml HKPG (d, e and f). Cytokine production is expressed as the mean ± SD in pg/ml for TNFα (a & d), IL-1β (b & e) and IL-6 (c & f). Data displayed represents triplicate samples for n = 3 replicate experiments. Significant differences in cytokine production between activated M1 and M2 MΦs are indicated as *p<0.05, **p<0.01, ***P<0.001 and ns, not significant.</p

PG-LPS exhibits weak endotoxin activity in THP-1-derived macrophages.

<p>THP-1-derived M1-like (PMA) and M2-like (Vit D₃) MΦs were either unstimulated (control) or stimulated with 100 ng/ml PG-LPS, 100 ng/ml <i>E. coli</i> K12 LPS (TLR4) or 10 µg/ml LTA (TLR2) for 18 hours. Endotoxin activity was investigated by TNFα secretion, presented as the mean ± SD in pg/ml. Data displayed is representative of triplicate samples for n = 3 replicate experiments.</p

<i>P. gingivalis</i> differentially suppresses CD14^hi/lo M1 and M2 MΦ IL-10 production.

<p>CD14^lo M1 (a) CD14^hi M1 (b), CD14^lo M2 (c) and CD14^hi M2 (d) MΦ subsets were pre-stimulated (tolerised) with either 100 ng/ml PG-LPS (unshaded) or 1×10⁷ cells/ml HKPG (shaded) for 24 hours prior to stimulation with PG-LPS or HKPG and incubated for a further 18 hours (untolerised controls indicated in bold). Anti-inflammatory IL-10 cytokine production is expressed in pg/ml as the mean ± SD for the CD14^hi/lo M1 and M2 MΦ subsets. Data displayed represents triplicate samples for n = 3 replicate experiments. Significant effects compared to the un-tolerised stimulus controls (bold) for each MΦ subset are are indicated as*p<0.05, **p<0.01, ***p<0.001 and ns, not significant.</p

P-110 Research and innovation – the importance of collaboration

What did we do? In 2020/2021, the hospice established a research and innovation subcommittee bringing together experts from a range of organisations across Central Lancashire including our acute and community NHS trusts and local university. The overall aim was to progress the hospice’s desire to be more actively involved in research and innovation, to improve the care of patients and their families. Why did we do it? Whilst the hospice had been involved in research, this had historically been fragmented, with limited strategic or focussed direction. We had a desire to improve and recognised that this would only be achieved by bringing together expertise and collaboration. The importance of research and innovation to the improvement in care and experience of patients and families, is unquestioned, and is a core component of the hospice’s five-year plan. What have we achieved? A quarterly research and innovation subcommittee chaired by the Pro-Vice Chancellor from our local university, has been established. A robust governance framework has been developed. A research and innovation three-year strategy has been agreed. The subcommittee has overseen the delivery of the hospice’s Equity of Access project, funded by Hospice UK and the Masonic Charitable Foundation. The hospice has been selected as a recruitment site for an NIHR portfolio study. A collaborative PhD, between the hospice and university has been developed and commenced, focused on how technology can support patient care. Agreed to support a PhD student undertaking a project exploring biophilic design. Confirmed support for a service evaluation project examining patient/carer/family feedback in palliative and end of life care. Monitoring of research related budgets. What are our plans for the future? The subcommittee is a formal part of the hospice governance structure. We will continue to progress the research agenda to deliver our strategy including developing a research dashboard, exploring opportunity for further doctoral students and developing our research infrastructure