Impaired surface expression of Transient Receptor Potential Melastatin 2 and 3 ion channels lowers Natural Killer Cell Cytotoxic Activity in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome

Myalgic Encephalomyelitis/ Chronic Fatigue Syndrome (ME/CFS) is a disabling condition characterised by unexplained chronic fatigue that is associated with immune, neurological (including autonomic), musculoskeletal, cardiovascular and gastrointestinal symptoms [1, 2]. Currently, accurate diagnosis remains challenging in the absence of a clinical or laboratory test. Although the aetiology of ME/CFS remains undefined, a significant reduction in natural killer (NK) cell cytotoxicity is consistently reported in ME/CFS patients compared with healthy controls (HC) [3-6]. NK cells are effector lymphocytes of the innate immune system principally responsible for recognising and responding to pathogen invasion [7]. Approximately 90% of peripheral NK cells are CD56DimCD16+ which are highly cytotoxic and kill infected, tumour or ‘missing self’ cells through cytotoxic processes [8]. Conversely, the CD56BrightCD16Dim/- subset is responsible for immunosurveillance and cytokine production [9]. Importantly, NK cells require calcium (Ca2+) to regulate various cellular functions, such as cell differentiation, cell division, apoptosis, transcription, and cytotoxicity [10]. Transient Receptor Potential (TRP) channels are a group of unique ion channels whereby majority are highly selective to Ca2+ [11]. Functionally, TRP ion channels regulate “threat” stimuli, such as pain, thermosensation, mechanosensation, pathogens, and chemicals, via sensory transduction pathways. TRPM2 and TRPM3 are cation channels ubiquitously expressed throughout the human body and expressed in almost all cell types, such as NK cells. Both TRPM members are highly permeable to Ca2+, therefore are critical regulators of Ca2+-dependent pathways, such as NK cell cytotoxicity. TRPM2 is activated by adenine dinucleotides (ADPR, cADPR, NAADP, ꞵ-NAD), reactive oxygen species (hydrogen peroxide and OH-), and intracellular ([Ca2+]i). Conversely, TRPM3 is potently activated by neural steroids, such as pregnenolone sulphate (PregS) and nifedipine. Upon stimulation, TRPM2 and TRPM3 ion channels trigger a rapid influx of Ca2+ influx and rise in [Ca2+]i, which subsequently facilitates intracellular pathways for Ca2+ release from cellular organelles. Recently, TRPM2 has emerged as a key receptor in mediating Ca2+-induced anti-tumour activity in mouse NK cells via synergistic activation with CD38. CD38 generates Ca2+ mobilizing secondary messengers, such as ADPR, to activate TRPM2. Rah et al., determined inhibition of sustained tumour-induced Ca2+ signals and degranulation in mouse NK cells following 8-bromoadenosine diphosphoribose (8-Br-ADPR) treatment [12]. Moreover, N6-Benzoyladenosine-3′,5′-cyclic monophosphate (N6-Bnz-cAMP) significantly increased intracellular ADPR, however was inhibited in Ca2+-free conditions [12]. Currently, no in vitro studies have examined the phenotype and function of TRPM2 ion channels on human NK cell subsets, notably in ME/CFS research. Conversely, five single nucleotide polymorphisms in NK cells associated with TRPM3 have been previously identified in ME/CFS patients [13]. Reduced TRPM3 surface expression and impaired Ca2+ influx has furthermore been identified on NK cells in ME/CFS patients [14]. Given TRPM2 and TRPM3 are both critical regulators for Ca2+ signalling in NK cells, the overall aim of this thesis was to investigate the role of TRPM2 and TRPM3 in mediating NK cell cytotoxicity to identify a potential mechanism of reduced NK cell cytotoxic activity in ME/CFS patients. Study one aimed to develop an in vitro methodology to characterise TRPM2 and CD38 surface expression on NK cell subsets using an antibody that has not been previously used with flow cytometry. Applying this optimised methodology, study two aimed to quantify TRPM2 and CD38 surface expression on NK cell subsets at baseline and post in vitro drug treatments (N6-Bnz-cAMP and 8-Br-ADPR) in ME/CFS patients and HC. NK cell cytotoxicity was furthermore measured at baseline and post in vitro drug treatments (N6-Bnz-cAMP and 8-Br-ADPR) between groups. Lastly, study three aimed to examine the clinical presentation in a moderate-severe ME/CFS group, as well as measure NK cell cytotoxicity post in vitro drug treatment with TRPM3 agonists, PregS, nifedipine and ononetin, in ME/CFS patients and HC. Age and sex matched HC were included in study one. Age and sex-matched ME/CFS patients meeting the Canadian Consensus Criteria (CCC) and HC were included in studies two and three. All participants donated 85ml of whole blood and peripheral NK cells were isolated. TRPM2 and CD38 surface expression was measured on CD56DimCD16+ and CD56BrightCD16Dim/- subsets, as well as NK cell cytotoxicity at baseline and post in vitro drug treatments by flow cytometry. Drug treatments included: interleukin-2, N6-Bnz-cAMP, 8-Br-ADPR, PregS, nifedipine and ononetin. Study one determined 1:50 as the optimal primary TRPM2 antibody dilution following a two-hour incubation period. TRPM2 surface expression with and without CD38 co-expression significantly increased between 1:300 and 1:50 primary TRPM2 antibody dilutions following a two-hour incubation period on both CD56DimCD16+ and CD56BrightCD16Dim/- NK cell subsets. On the CD56DimCD16+ subset only, TRPM2 and CD38 surface expression also significantly increased at 1:50 compared with 1:100. Moreover, TRPM2 surface expression significantly decreased between 1:50 and 1:5 TRPM2 antibody dilution following a two-hour incubation period. This significant decrease highlights the high-dose hook effect, whereby the highly concentrated 1:5 antibody dilution saturated both capture and detection TRPM2 antibodies. Study two identified a significant overexpression of the TRPM2 ion channel on NK cell subsets in ME/CFS patients compared with HC. No significant differences in NK cell cytotoxicity were observed between or within groups post N6-Bnz-cAMP and 8-Br-ADPR drug treatments. Lastly, study three revealed no signficiant differences in NK cell cytotoxicity post PregS and nifedipine drug treatments, as well as subsequent blocking with ononentin in both groups. In both Study 2 and Study 3, viral infections and various clinical ME/CFS symptoms were significantly associated with reduced NK cell cytotoxicity in ME/CFS patients. Associations included: pain, cognitive difficulties, sleep disturbances, sensory impairments, thermostatic instability and gastrointestinal disturbances, possibly involving TRPM2 and TRPM3. In conclusion, the results of this thesis are the first to develop a novel and optimal in vitro methodology to measure TRPM2 and CD38 surface expression on human NK cell subsets using flow cytometry. This thesis is also the first to report overexpressed TRPM2 ion channels on NK cell subsets in ME/CFS patients. Oxidative stress induced by viral infections is hypothesised to cause this overexpression in TRPM2 ion channels in ME/CFS patients as previously reported. Overexpressed TRPM2 ion channels may cause mitochondrial dysfunction, cellular death, DNA damage, and disruption to MAPK pathways following uncontrolled increases in [Ca2+]i. Collectively, these processes interfere with downstream Ca2+-dependent pathways, such as NK cell cytotoxicity, which was found to be significantly reduced at baseline in ME/CFS patients compared to HC. The drug-treated NK cell cytotoxicity results may reflect the sensitivity of the cytotoxic assay to capture the TRPM2 and TRPM3 drug-modulatory effects on NK cell cytotoxicity. Given the drugs were incubated in the media and cells for more than 24 hours, the TRPM2 and TRPM3 ion channels may have undergone repetitive activation and inhibition cycles. Consequently, this may have activated and caused sustained long-term Ca2+-dependent pathways which may have potentially resulted in disrupted gene expression, irreversible cellular death and the development of NK cell hyporesponsiveness. Furthermore, a primary limitation with TRPM2 is the lack of potent and specific pharmacological tools. Consequently, the TRPM2 signals may have been reduced or lost, subsequently modulating activation of downstream NK cell cytotoxic processes, such as the extracellular signal-regulated protein kinase 1/2 and mitogen-activated protein kinase pathways. Taken together, this thesis warrants the identification of additional experiments with a more appropriate time-sensitivity to capture the pharmacological effects of specific cellular mechanisms of interest, as well as the identification and development of more potent, specific, and non-toxic pharmacological tools targeting TRPM2. Additional rationales include co-localisation of between TRPM2 and CD38 and the involvement of TRPM2 and TRPM3 spliced isoforms. A common feature shared amongst the significant associations between reduced NK cell cytotoxicity clinical ME/CFS symptoms is the high expression and functional activity of TRPM2 and TRPM3 in the CNS, which functions as the control centre for these physiological systems. However, additional quantitative tests examining these clinical functions, such as nociceptive pain and thermoregulation, are required to definitvely associate the possible roles of TRPM2 and TRPM3 activity and the unique clinical presentation of ME/CFS. Interestingly, positive correlations were determined between reduced NK cell cytotoxicity and overexpressed TRPM2 ion channels on both NK cell subsets within the ME/CFS group. ROC analyses also revealed diagnostic potential for reduced NK cell cytotoxicity and overexpressed TRPM2 ion channels in ME/CFS patients. Collectively, these results highlight a relationship between TRPM2 and reduced NK cell cytotoxicity in ME/CFS. Therefore, furthur investigations in this vital area of ME/CFS research will assist in the validation of TRPM2 and TRPM3 as potential biological markers to further understand the unique pathomechanism of ME/CFS and facilitate the development of targeted therapeutic interventions to improve the quality of life of ME/CFS patients.

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