Award details

Biosensor based approach to measure release of Nociceptin/Orphanin FQ from live single immune cells and consequences for immune cell function.

Reference	BB/N000188/1
Principal Investigator / Supervisor	Professor David Lambert
Co-Investigators / Co-Supervisors	Dr Jon Willets
Institution	University of Leicester
Department	Cardiovascular Sciences
Funding type	Research
Value (£)	338,432
Status	Completed
Type	Research Grant
Start date	01/01/2016
End date	31/12/2018
Duration	36 months

Abstract

The receptor for nociceptin/orphanin FQ (N/OFQ) or NOP is expressed (mRNA) on all immune cells where there is limited evidence for N/OFQ modulation of immune function. N/OFQ is produced from pre-pro-N/OFQ or ppNoc and we have detected mRNA for ppNoc in monocytes, lymphocytes and eosinophils. We have a Chinese Hamster Ovary clone expressing chimeric Galpha-i/q protein enabling Gi to increase Calcium; this can be measured fluorimetrically. This cell line is co-transfected with NOP allowing NOP activation increase Calcium; a biosensor for N/OFQ. If we overlay Galpha-i/q cells with polymorphonuclear leucocytes and stimulate them to degranulate we can measure N/OFQ release at the single cell level for the first time. We have two hypotheses; 1-Leucocytes differentially release N/OFQ and we will use our biosensor system to measure this the single cell level; 2-NOP activation will modulate leucocyte function at the cellular level and we will use intracellular signalling pathways and migration as readouts. We will develop and characterise this NOP-Galpha-i/q chimera based biosensor in confocal microscopy. Blood will be separated into monocytes, B/T lymphoctyes and basophil, neutrophil and eosinophil granulocytes and assessed for N/OFQ release. We will assess antagonist sensitivity to confirm NOP as the target and explore Calcium dependence of release. Cellular content of N/OFQ peptide will be measured by immunofluoresence. We will characterise the effects of immune cell NOP activation on ERK1/2 and JNK using Western blot along with apoptosis (cytochrome c and cleaved caspase 3). We will examine the effects of NOP activation on RhoGTPase using the Active RHO pull down/detection kit as this has been implicated in migration. We will also measure cytokine release from cell populations in the absence and presence of N/OFQ using Luminex xMAP technology. We will use transwell migration assays to assess autocrine and paracrine control of migration and the effects of NOP activation.

Summary

Context: Classical opioid receptors are classified as MOP(mu), DOP(delta) and KOP(kappa). In addition to these three receptor types there is a fourth non-classical type, the receptor for nociceptin/orphaninFQ (N/OFQ) or NOP. The peptide N/OFQ is produced from a precursor pre-pro-N/OFQ (ppNoc). Opioids cause immune suppression but the mechanisms are poorly understood; in particular for the N/OFQ-NOP system. In this regard all immune cells we have tested to date express mRNA for NOP but variably express mRNA for N/OFQ. Little is known about the release of N/OFQ from immune cells and nothing at the single cell level. We have a Chinese Hamster Ovary (CHO) cell clone that expresses a chimeric Galpha-i/q protein enabling Gi coupled receptors to increases in intracellular Calcium; this can be measured fluorimetrically. This cell line is co-transfected with NOP; effectively allowing receptor NOP activation to be monitored as a Calcium signal; a biosensor for N/OFQ. If we overlay Galpha-i/q cells with polymorphonuclear leucocytes and stimulate them to degranulate with fMLP we have been able to measure N/OFQ release at the single cell level for the first time. This release was sensitive to NOP antagonists but not purinergic antagonists. There are two hypotheses to our application; 1-Leucocytes differentially release N/OFQ and we will develop a novel cell based Galpha-i/q biosensor system to measure this at the single cell level; 2-NOP activation by N/OFQ will modulate leucocyte function at the cellular physiological level and we will use the intracellular signalling pathways and migration as readouts. Aims and objectives: We will develop and characterise a Galpha-i/q chimera based biosensor assay for use with cells expressing recombinant human NOP in confocal microscopy. Volunteer human blood will be separated into individual immune cell populations (monocytes, B and T lymphoctyes and basophil, neutrophil and eosinophil granulocytes) with each assessed for release capability. Based on preliminary data showing differential ppNoc expression we predict that of the granulocyte population, eosinophils will be the source of N/OFQ. In addition we predict that monocytes and lymphocytes will also release N/OFQ and that we can measure this from single cells. We will assess antagonist sensitivity to confirm NOP as the target, explore Calcium dependence of the release process and confirm each cell immune cell type by immunofluoresence. Cellular content of N/OFQ peptide will also be measured by immunofluoresence with specific N/OFQ antibodies. Of note, there are no NOP reliable antibodies for use in Western blotting. We will characterise the effects of immune cell NOP activation with peptide and non-peptide agonists by measuring ERK1/2 phosphorylation and JNK using Western blot. Activation of ERK1/2 has been linked to apoptosis so we will also measure cytochrome c and cleaved caspase 3 as we have done previously in neuronal cells. As Rho GTPases are linked with cell migration and we will be assessing this we will examine the effects of NOP activation on Rho GTPase using the Active RHO pull down and detection kit. We will also measure the cytokine release profile from cell populations in response to degranulation stimuli in the absence and presence of N/OFQ using Luminex xMAP technology and the Human 25-Plex panel. We will use transwell migration assays to assess autocrine and paracrine control of migration and the effects of NOP activation. Applications/Benefits: Data from this project will provide: (1) a novel technique for measuring single-cell release, (2) the first detailed release profile of N/OFQ from immune cells, currently lacking in the literature and (3) detailed information of the consequences of that release at a biochemical and functional level. Furthermore, we believe that these experimental techniques and data will have applicability to other cells and systems (e.g. neuronal transmitter release).

Impact Summary

Who will benefit from this research ? List beneficiaries. 1. Those working on the effects of opioids, the immune system and much more specifically the immune effects of opioids. 2. Pharmaceutical companies with activity in pain/opioids (e.g., GSK, Grunenthal and Pfizer). 3. Medical practitioners; in particular those looking after patients taking chronic opioids and those specializing in palliative care. 4. Lay public with an interest in drug development, drug (opioid) abuse and immune function. How will they benefit ? 1. Opioid/immunology researchers. We will provide new information on immune cell nociceptin release and its effects on immune function. We will provide a new way of measuring single release using a biosensor based approach. This approach will be of use to others in related fields, for example those wanting to measure release from neurones. The timescale for this activity will be from around 6 months into and continue throughout the project. 2. Pharmaceutical industry. As part of the drug development process assessment of adverse drug reactions is essential. A better understanding of immune suppression is important in opioid development and new ways of screening for effects is of interest. An indicative timescale here would be an approach mid way through the second year. 3. Medical practitioners. There are a number of opioids that can be used long term and in palliative care. Moreover, there is advanced development of ligands for the nociceptin receptor (e.g., cebranopadol; a mixed agonist also acting at mu receptors). A better understanding of drug specific immune suppression will aid in drug selection for particular patients (personalised medicine). 4. Lay public will benefit from our outreach strategy to (i) demystify opioids, opioid abuse and opiophobia and (ii) educate on drug development. Time frames for items 3 and 4 would be towards the end of the first and second years in the form of talks, web materials and information booklets. In this regard we will remain responsive to changes in the field. The named post-doc Mr Bird will benefit from science specific skills but also from more generic skills such as; (i) public engagement, (ii) web design, (iii) use of social media in an educational context.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	Immunology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

I accept the terms and conditions of use (opens in new window)

export PDF file

back to list new search