title: Implications of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome on quality of life and the role of transient receptor potential melastatin 3 ion channels on natural killer cells in the pathomechanism of illness
Creators: name: Eaton-Fitch, Natalie R
Publisher: name: Griffith University
Year: 2022
Subjects: subject: Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS)
subject: symptom
subject: quality of life (QoL)
subject: Immunological dysfunction
subject: natural killer (NK) cell
subject: Australian

description: Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a serious disorder of unknown aetiology with an abstruse pathomechanism. Diagnosis is often delayed due to the lack of laboratory tests and specific diagnostic criteria as well as the lack of effective treatments that heightens the burden of illness. ME/CFS is characterised by post-exertional malaise and neuroimmune exhaustion, accompanied by symptoms that are neurological, immunological, endocrinological, gastrointestinal, and genitourinary in nature. To date, the cause and origin remain ambiguous, hence patients often try different treatment options to improve health, but without much success. The burden of ME/CFS on quality of life (QoL) is a key component of this thesis, therefore, the first aim was to determine the impact of ME/CFS on QoL. For the first phase of this thesis, using an online survey, self-reported data was collected from 480 individuals diagnosed with ME/CFS (46.0 ± 12.3 years). A multivariate linear regression was used to determine the correlation between ME/CFS symptom presentation with QoL. Health-related QoL (HRQoL) was significantly impaired in ME/CFS patients across all eight SF-36 domains when compared with Australian general population norms obtained from the Australian Bureau of Statistics (p&lt;0.001). ME/CFS patients reported the lowest SF-36 scores for physical role of illness on limitations (4.1 ± 15.1). Lower QoL scores in limitations due to physical functioning were associated with gender (p&lt;0.001, adjusted R2=0.236), high body mass index (BMI) (p=0.015, adjusted R2=0.236), unemployment (p&lt;0.001, adjusted R2=0.236), cognitive difficulties (p=0.014, adjusted R2=0.236), sensory disturbances (p&lt;0.001, adjusted R2=0.236) and cardiovascular symptoms (p&lt;0.001, adjusted R2=0.236). Low scores for limitations due to pain were associated with high BMI (p=0.008, adjusted R2=0.497), flu-like symptoms (p=0.037, adjusted R2=0.497) and body temperature intolerances (p=0.035, adjusted R2=0.497), as well as frequent visits to their general practitioner (p=0.035, adjusted R2=0.497). Reduced well-being QoL scores were associated with smoking status (p=0.013, adjusted R2=0.155), psychiatric comorbidity (p&lt;0.001, adjusted R2=0.155), sleep disturbances (p&lt;0.001, adjusted R2=0.155) and gastrointestinal upset (p=0.038, adjusted R2=0.155). Immunological dysfunction is a key aspect of the presentation of ME/CFS, the significantly reduced natural killer (NK) cell cytotoxicity has been consistently reported. However, this the consistence of impaired NK cell cytotoxicity was yet to be validated through systematic review. In phase II, a systematic review was completed to investigate NK cell cytotoxicity in ME/CFS patients compared with HC. Impaired NK cell cytotoxicity was a consistent immunological feature in ME/CFS patients, and this systematic review confirmed the validity of NK cell cytotoxicity as a reliable vector for research in ME/CFS. Therefore, NK cells were used as a cell model for ME/CFS research in phases III and IV of this thesis. NK cell cytotoxicity heavily relies on long-term sustained influx of calcium (Ca2+) to successfully activate function through intracellular signalling proteins, polarisation of cytolytic granules and release of cytolytic proteins. Therefore, addition focus of this thesis was to investigation Ca2+ mobilisation and characterise transient receptor potential melastatin 3 (TRPM3) channels. TRPM3 ion channels are highly permeable to Ca2+, and have critical roles in pain perception, thermosensation and inflammation. In the laboratory, TRPM3 can be activated using pregnenolone sulfate (PregS) and inhibited using ononetin. Recently, treatment of NK cells in vitro using naltrexone hydrochloride (NTX) has restored TRPM3 function in ME/CFS patients. It is hypothesised that some ME/CFS subsets may be the result of ion channelopathy, and that this research will contribute to the understanding of TRPM3 and Ca2+ mobilisation to the mechanism of ME/CFS. NK cells were isolated from whole blood of participants using negative immunomagnetic selection. In phase III, NK cells were isolated from n=15 ME/CFS patients (43.93±2.94 years, 60% female) and n=15 healthy controls (HC) (44.2±3.14 years, 60% female). The co-localisation of TRPM3 with Ca2+ signalling protein, phosphatidylinositol 4,5-bisphosphate (PIP2), after regulation with TRPM3 specific drugs including PregS and ononetin, was investigated using immunofluorescence. Finally, in phase IV, NK cells were isolated from n=10 ME/CFS patients (43.9±10.71 years, 90% female) and n=10 HC (44.1±10.39, 90% female) to investigate TRPM3-dependent Ca2+ influx using live-cell immunofluorescent imaging. Inferential analysis of co-localisation between HC and ME/CFS patients revealed a significant reduction in co-localisation of TRPM3 with PIP2 in ME/CFS patients under control conditions (p=0.0021). Interestingly, a significant increase in co-localisation of TRPM3 with PIP2 was reported in ME/CFS patients compared with HC following ononetin (p=0.0408). NK cell cytotoxicity was significantly reduced in ME/CFS patients compared with HC at baseline (p=0.0362). Overnight interleukin-2 (IL-2) stimulation significantly enhanced NK cell cytotoxicity in both HC and ME/CFS patients (p&lt;0.0001). However, no significant difference was reported in NK cell cytotoxicity within groups following pharmacological treatment supplemented with IL-2. TRPM3-dependent Ca2+ influx was determined following stimulation with PregS using three measurements: slope, half-time response (T1/2) and amplitude. Baseline Ca2+ influx was determined. The amplitude and T1/2 response were significantly reduced in ME/CFS patients compared with HC (p&lt;0.0001). Ca2+ influx measurements for slope (p&lt;0.0001), amplitude (p&lt;0.0001) and T1/2 (p=0.0123) were significantly lower in ME/CFS patients compared with HC following overnight stimulation with IL-2. Interestingly, overnight treatment of isolated NK cells with NTX restored TRPM3-dependent Ca2+ influx in ME/CFS patients. There was no longer a significant difference between groups for slope and T1/2 response. A significant increase in amplitude was reported in ME/CFS patients compared with HC (p&lt;0.0001). In conclusion, ME/CFS is a serious condition and associated with reduced HRQoL emphasising the significant burden of illness, the importance of renewed public health policy, and increased research funding for ME/CFS patients. This thesis was the first to report on the significant association of low QoL scores with symptom presentation and severity in a large cohort of Australian ME/CFS patients. An example was the significant association found between reduced QoL and flu-like symptoms. ME/CFS is a multifactorial condition, and a key feature is immunological dysfunction, specifically reduced NK cell cytotoxicity. However, as ME/CFS is an ambiguous and multifactorial condition, the use of an appropriate model is vital for research. This thesis reports novel findings to support and confirm NK cell cytotoxicity is a consistent finding and model for research into the pathomechanism of ME/CFS. While TRPM3 ion channel dysfunction is well documented using whole-cell patch clamp, the intracellular signalling pathways involved in TRPM3 dysfunction is elusive. This research reveals, for the first time, the potential role of PIP2 in TRPM3 ion channel dysfunction. The potential crosstalk of signalling proteins involved in TRPM3, PIP2 and IL-2 signalling pathways in lymphocytes provides a foundation for future research in ME/CFS. The characterisation of TRPM3 ion channels provides an important target for not only elucidating the pathomechanism of ME/CFS, but also provides a potential therapeutic target. By targeting TRPM3 ion channels through pharmacotherapeutic modalities, namely NTX, this thesis reported novel findings for the restoration of intracellular Ca2+ influx in NK cells of ME/CFS patients using immunofluorescent imaging. TRPM3 is widely expressed in many cell and tissue types throughout the human body and by targeting TRPM3 ion channels with NTX, this may improve the multisystemic clinical presentation of ME/CFS patients, which may increase the QoL of these patients. Importantly, the use of a reliable and accessible cellular model for undertaking research into the pathomechanism of ME/CFS may advance research across disciplines and allow for the discovery of pharmacotherapeutic interventions to improve HRQoL.