COVID-19 related myocardial injury is associated with immune dysregulation in symptomatic patients with cardiac MRI abnormalities.

AIMS: While acute cardiovascular complications of COVID-19 are well-described, less is known about longer-term cardiac sequelae. For many individuals with cardiac signs or symptoms arising after COVID-19 infection, the aetiology remains unclear. We examined immune profiles associated with magnetic resonance imaging (MRI) abnormalities in patients with unexplained cardiac injury after COVID-19. METHODS AND RESULTS: Twenty-one participants (mean age 47 [SD 13] years, 71% female) with long COVID (n=17), raised troponin (n=2), or unexplained new-onset heart failure (n=2), who did not have pre-existing heart conditions or recent steroid/immunosuppression treatment were enrolled a mean 346 (SD 191) days after COVID-19 infection in a prospective observational study. Cardiac MRI and blood sampling for deep immunophenotyping using mass cytometry by time of flight and measurement of proteomic inflammatory markers was performed. Nine of 21 (43%) participants had MRI abnormalities (MRI(+)), including non-ischaemic patterns of late gadolinium enhancement and/or visually overt myocardial oedema in 8 people. One patient had mildly impaired biventricular function without fibrosis or oedema, and 2 had severe left ventricular impairment. MRI(+) individuals had higher blood CCL3, CCL7, FGF-23 and CD4 Th2 cells, and lower CD8 T effector memory (TEM) cells, than MRI(-). Cluster analysis revealed lower expression of inhibitory receptors PD1 and TIM3 in CD8 TEM cells from MRI(+) patients than MRI(-) patients, and functional studies of CD8 T αß cells showed higher proportions of cytotoxic granzyme B+ secreting cells upon stimulation. CD8 TEM cells and CCL7 were the strongest predictors of MRI abnormalities in a LASSO regression model (composite AUC 0.96, 95%CI 0.88-1.0). CCL7 was correlated with diffuse myocardial fibrosis/oedema detected by quantitative T1 mapping (r=0.47, p=0.04). CONCLUSION: COVID-19 related cardiac injury in symptomatic patients with non-ischaemic myocarditis-like MRI abnormalities is associated with immune dysregulation, including decreased peripheral CD8 TEM cells and increased CCL7, persisting long after the initial infection.

Opioids and cancer survival: are we looking in the wrong place?

There is a controversial narrative in the anaesthetic literature suggesting that anaesthetic technique (including opioids) may be detrimental to survival after tumour resection. The initial observations were retrospective. Several prospective studies are ongoing; one in breast cancer has reported no adverse outcome. The evidence for an effect of opioids stems from three pieces of information: (1) opioids depress the immune system, (2) opioids potentially promote angiogenesis, and (3) opioids potentially support tumour growth. Although the evidence for (2)/(3) is unclear, combinations of these effects are beneficial to tumours and potentially promote metastatic reseeding. Accepted wisdom suggests that opioid effects are driven by opioid receptor activation but the presence of opioid receptors on immune cells for example is unlikely. Immune cells, vascular endothelium and a range of tumour cells express Toll-like receptor 4 (TLR4) receptors (for Gram-negative bacterial wall components), and there is growing evidence for opioids interacting with this alternative receptor; and for some there is paradoxical naloxone sensitivity. Is the focus on opioid receptors and cancer the wrong target? TLR4 receptor activation produces immune activation, stimulates angiogenesis, and supports tumour survival. We know that some opioids are more immune suppressive than others (there is no such comparative information for angiogenesis and tumour survival); this may correlate with TLR4 activation. If there are clusters of opioids that have more opioid than TLR4 profiles and vice versa, this may influence outcome. If this is the case, then evidence-based advice could be given for perioperative use in the oncology-anaesthesia setting.

Nociceptin/Orphanin FQ receptor expression in primary human umbilical vein endothelial cells is not regulated by exposure to breast cancer cell media or angiogenic stimuli.

Background: Opioid receptors are naloxone-sensitive (MOP [mu: µ], DOP [delta: δ], and KOP [kappa: κ]) and naloxone-insensitive Nociceptin/Orphanin FQ (N/OFQ) peptide receptor (NOP). Clinically, most opioid analgesics target MOP. Angiogenesis is the formation of new blood vessels and involves endothelial cell activation, proliferation, and migration. The effect of opioids on this process is controversial with no data for NOP receptor ligands. Methods: We used patient-derived human umbilical vein endothelial cells (HUVECs) treated with media from the Michigan Cancer Foundation-7 (MCF-7) breast cancer cells or vascular endothelial growth factor (VEGF; 10 ng ml-1) and fibroblast growth factor (FGF; 10 ng ml-1) as angiogenic stimuli. We have measured (i) NOP/MOP messenger RNA, (ii) receptor protein using N/OFQATTO594 and DermorphinATTO488 as fluorescent probes for NOP and MOP, and (iii) NOP/MOP function in a wound healing assay (crude measure of migration that occurs during angiogenesis). Results: HUVEC lines from 32 patients were used. Using all 32 lines, mRNA for NOP but not MOP was detected. This was unaffected by media from MCF-7 cells or VEGF/FGF. There was no binding of either N/OFQATTO594(NOP) or DermorphinATTO488(MOP) in the absence or presence of angiogenic stimuli (six lines tested). In the absence of MOP mRNA, this was expected. Whilst MCF-7 conditioned medium (not VEGF/FGF) reduced wound healing per se (14 lines tested), there was no effect of N/OFQ (NOP ligand) or morphine (MOP ligand). Conclusions: Media from MCF-7 breast cancer cells or VEGF/FGF as angiogenic stimuli did not influence NOP translation into receptor protein. MOP was absent. In the absence of constitutive or inducible MOP/NOP, there was no effect on wound healing as a measure of angiogenesis.

Fluorescent opioid receptor ligands as tools to study opioid receptor function.

Opioid receptors are divided into the three classical types: MOP(µ:mu), DOP(δ:delta) and KOP(κ:kappa) that are naloxone-sensitive and an additional naloxone-insensitive nociceptin/orphanin FQ(N/OFQ) peptide receptor(NOP). Studies to determine opioid receptor location and turnover variably rely on; (i) measuring receptor mRNA, (ii) genetically tagging receptors, (iii) labelling receptors with radioligands, (iv) use of antibodies in immunohistochemistry/Western Blotting or (v) measuring receptor function coupled with the use of selective antagonists. All have their drawbacks with significant issues relating to mRNA not necessarily predicting protein, poor antibody selectivity and utility of radiolabels in low expression systems. In this minireview we discuss use of fluorescently labelled opioid receptor ligands. To maintain the pharmacological properties of the corresponding parent ligand fluorescently labelled ligands must take into account fluorophore (brightness and propensity to bleach), linker length and chemistry, and site to which the linker (and hence probe) will be attached. Use of donor and acceptor fluorophores with spectral overlap facilitates use in FRET type assays to determine proximity of ligand or tagged receptor pairs. There is a wide range of probes of agonist and antagonist nature for all four opioid receptor types; caution is needed with agonist probes due to the possibility for internalization. We have produced two novel ATTO based probes; DermorphinATTO488 (MOP) and N/OFQATTO594 (NOP). These probes label MOP and NOP in a range of preparations and using N/OFQATTO594 we demonstrate internalization and ligand-receptor interaction by FRET. Fluorescent opioid probes offer potential methodological advantages over more traditional use of antibodies and radiolabels.

Evaluation of [Cys(ATTO 488)8]Dermorphin-NH2 as a novel tool for the study of µ-opioid peptide receptors.

The µ-opioid peptide (MOP) receptor is a member of the opioid receptor family and an important clinical target for analgesia. Measuring MOP receptor location and tracking its turnover traditionally used radiolabels or antibodies with attendant problems of utility of radiolabels in whole cells and poor antibody selectivity. To address these issues we have synthesized and characterised a novel ATTO488 based fluorescent Dermorphin analogue; [Cys(ATTO 488)8]Dermorphin-NH2 (DermATTO488). We initially assessed the binding profile of DermATTO488 in HEK cells expressing human MOP and CHO cells expressing human MOP, δ-opioid peptide (DOP), κ-opioid peptide (KOP) and Nociceptin/Orphanin FQ peptide (NOP) receptors using radioligand binding. Functional activity of the conjugated peptide was assessed by measuring (i) the ability of the ligand to engage G-protein by measuring the ability to stimulate GTPγ[35S] binding and (ii) the ability to stimulate phosphorylation of ERK1/2. Receptor location was visualised using confocal scanning laser microscopy. Dermorphin and DermATTO488 bound to HEKMOP (pKi: 8.29 and 7.00; p<0.05), CHOMOP (pKi: 9.26 and 8.12; p<0.05) and CHODOP (pKi: 7.03 and 7.16; p>0.05). Both ligands were inactive at KOP and NOP. Dermorphin and DermATTO488 stimulated the binding of GTPγ[35S] with similar pEC50 (7.84 and 7.62; p>0.05) and Emax (1.52 and 1.34fold p>0.05) values. Moreover, Dermorphin and DermATTO488 produced a monophasic stimulation of ERK1/2 phosphorylation peaking at 5mins (6.98 and 7.64-fold; p>0.05). Finally, in confocal microscopy DermATTO488 bound to recombinant MOP receptors on CHO and HEK cells in a concentration dependent manner that could be blocked by pre-incubation with unlabelled Dermorphin or Naloxone. Collectively, addition to ATTO488 to Dermorphin produced a ligand not dissimilar to Dermorphin; with ~10fold selectivity over DOP. This new ligand DermATTO488 retained functional activity and could be used to visualise MOP receptor location.