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Transient receptor potential melastatin 2 channels are overexpressed in myalgic encephalomyelitis/chronic fatigue syndrome patients.

ABSTRACT:

Background

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is hallmarked by a significant reduction in natural killer (NK) cell cytotoxicity, a mechanism tightly regulated by calcium (Ca²⁺). Interestingly, interleukin-2 (IL-2) increases NK cell cytotoxicity. Transient receptor potential melastatin 2 (TRPM2) ion channels are fundamental for Ca²⁺ signalling in NK cells. This pilot investigation aimed to characterise TRPM2 and CD38 surface expression in vitro on NK cells in ME/CFS patients. This investigation furthermore examined the pharmaceutical effect of 8-bromoadenosine phosphoribose (8-Br-ADPR) and N₆-Benzoyladenosine-3',5'-cyclic monophosphate (N₆-Bnz-cAMP) on TRPM2 and CD38 surface expression and NK cell cytotoxicity between ME/CFS and healthy control (HC) participants.

Methods

Ten ME/CFS patients (43.45 ± 12.36) and 10 HCs (43 ± 12.27) were age and sex-matched. Isolated NK cells were labelled with fluorescent antibodies to determine baseline and drug-treated TRPM2 and CD38 surface expression on NK cell subsets. Following IL-2 stimulation, NK cell cytotoxicity was measured following 8-Br-ADPR and N₆-Bnz-cAMP drug treatments by flow cytometry.

Results

Baseline TRPM2 and CD38 surface expression was significantly higher on NK cell subsets in ME/CFS patients compared with HCs. Post IL-2 stimulation, TRPM2 and CD38 surface expression solely decreased on the CD56^DimCD16⁺ subset. 8-Br-ADPR treatment significantly reduced TRPM2 surface expression on the CD56^BrightCD16^Dim/- subset within the ME/CFS group. Baseline cell cytotoxicity was significantly reduced in ME/CFS patients, however no changes were observed post drug treatment in either group.

Conclusion

Overexpression of TRPM2 on NK cells may function as a compensatory mechanism to alert a dysregulation in Ca²⁺ homeostasis to enhance NK cell function in ME/CFS, such as NK cell cytotoxicity. As no improvement in NK cell cytotoxicity was observed within the ME/CFS group, an impairment in the TRPM2 ion channel may be present in ME/CFS patients, resulting in alterations in [Ca²⁺]_i mobilisation and influx, which is fundamental in driving NK cell cytotoxicity. Differential expression of TRPM2 between NK cell subtypes may provide evidence for their role in the pathomechanism involving NK cell cytotoxicity activity in ME/CFS.

SUBMITTER: Balinas C

PROVIDER: S-EPMC6891975 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundMyalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a severe multisystemic condition associated with post-infectious onset, impaired natural killer (NK) cell cytotoxicity and impaired ion channel function, namely Transient Receptor Potential Melastatin 3 (TRPM3). Long-term effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has resulted in neurocognitive, immunological, gastrointestinal, and cardiovascular manifestations recently recognised as post coronavirus disease 2019 (COVID-19) condition. The symptomatology of ME/CFS overlaps significantly with post COVID-19; therefore, this research aimed to investigate TRPM3 ion channel function in post COVID-19 condition patients.MethodsWhole-cell patch-clamp technique was used to measure TRPM3 ion channel activity in isolated NK cells of N = 5 ME/CFS patients, N = 5 post COVID-19 patients, and N = 5 healthy controls (HC). The TRPM3 agonist, pregnenolone sulfate (PregS) was used to activate TRPM3 function, while ononetin was used as a TRPM3 antagonist.ResultsAs reported in previous research, PregS-induced TRPM3 currents were significantly reduced in ME/CFS patients compared with HC (p = 0.0048). PregS-induced TRPM3 amplitude was significantly reduced in post COVID-19 condition compared with HC (p = 0.0039). Importantly, no significant difference was reported in ME/CFS patients compared with post COVID-19 condition as PregS-induced TRPM3 currents of post COVID-19 condition patients were similar of ME/CFS patients currents (p > 0.9999). Isolated NK cells from post COVID-19 condition and ME/CFS patients were resistant to ononetin and differed significantly with HC (p < 0.0001).ConclusionThe results of this investigation suggest that post COVID-19 condition patients may have impaired TRPM3 ion channel function and provide further evidence regarding the similarities between post COVID-19 condition and ME/CFS. Impaired TRPM3 channel activity in post COVID-19 condition patients suggest impaired ion mobilisation which may consequently impede cell function resulting in chronic post-infectious symptoms. Further investigation into TRPM3 function may elucidate the pathomechanism, provide a diagnostic and therapeutic target for post COVID-19 condition patients and commonalities with ME/CFS patients.

Project description:BACKGROUND:Chronic Fatigue Syndrome (CFS)/ Myalgic Encephalomyelitis (ME) is a debilitating disorder that is accompanied by reduced cytotoxic activity in natural killer (NK) cells. NK cells are an essential innate immune cell, responsible for recognising and inducing apoptosis of tumour and virus infected cells. Calcium is an essential component in mediating this cellular function. Transient Receptor Potential Melastatin 3 (TRPM3) cation channels have an important regulatory role in mediating calcium influx to help maintain cellular homeostasis. Several single nucleotide polymorphisms have been reported in TRPM3 genes from isolated peripheral blood mononuclear cells, NK and B cells in patients with CFS/ME and have been proposed to correlate with illness presentation. Moreover, a significant reduction in both TRPM3 surface expression and intracellular calcium mobilisation in NK cells has been found in CFS/ME patients compared with healthy controls. Despite the functional importance of TRPM3, little is known about the ion channel function in NK cells and the epiphenomenon of CFS/ME. The objective of the present study was to characterise the TRPM3 ion channel function in NK cells from CFS/ME patients in comparison with healthy controls using whole cell patch-clamp techniques. METHODS:NK cells were isolated from 12 age- and sex-matched healthy controls and CFS patients. Whole cell electrophysiology recording has been used to assess TRPM3 ion channel activity after modulation with pregnenolone sulfate and ononetin. RESULTS:We report a significant reduction in amplitude of TRPM3 current after pregnenolone sulfate stimulation in isolated NK cells from CFS/ME patients compared with healthy controls. In addition, we found pregnenolone sulfate-evoked ionic currents through TRPM3 channels were significantly modulated by ononetin in isolated NK cells from healthy controls compared with CFS/ME patients. CONCLUSIONS:TRPM3 activity is impaired in CFS/ME patients suggesting changes in intracellular Ca2+ concentration, which may impact NK cellular functions. This investigation further helps to understand the intracellular-mediated roles in NK cells and confirm the potential role of TRPM3 ion channels in the aetiology and pathomechanism of CFS/ME.

Project description:Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a seriously long-term and debilitating illness of unknown cause hallmarked by chronic pain and fatigue, memory and concentration impairment, and inflammation. ME/CFS hypothesis involves impaired Transient receptor potential melastatin 3 (TRPM3) ion channel function, affecting calcium signaling and Natural killer (NK) cell functions. Currently, substances called opioids, agonists of mu (?)-opioid receptors (?OR), are the strongest painkillers clinically available for people suffering from strong or long-lasting pain characteristic of ME/CFS. ?OR have been reported to specifically inhibit TRPM3 and to be expressed in immune cells where they play an immunomodulatory and immunosuppressive role. Naltrexone hydrochloride (NTX) acts as an antagonist to the ?OR thus negating the inhibitory function of this opioid receptor on TRPM3. Therefore, understanding the mechanism of action for NTX in regulating and modulating TRPM3 channel function in NK cells will provide important information for the development of effective therapeutic interventions for ME/CFS. Whole-cell patch-clamp technique was used to measure TRPM3 activity in Interleukin-2 (IL-2) stimulated and NTX-treated NK cells for 24 h on eight ME/CFS patients and 8 age- and sex-matched healthy controls, after modulation with a TRPM3-agonist, pregnenolone sulfate (PregS), NTX and a TRPM3-antagonist, ononetin. We confirmed impaired TRPM3 function in ME/CFS patients through electrophysiological investigations in IL-2 stimulated NK cells after modulation with PregS and ononetin. Importantly, TRPM3 channel activity was restored in IL-2 stimulated NK cells isolated from ME/CFS patients after incubation for 24 h with NTX. Moreover, we demonstrated that NTX does not act as an agonist by directly coupling on the TRPM3 ion channel gating. The opioid antagonist NTX has the potential to negate the inhibitory function of opioid receptors on TRPM3 in NK cells from ME/CFS patients, resulting in calcium signals remodeling, which will in turn affect cell functions, supporting the hypothesis that NTX may have potential for use as a treatment for ME/CFS. Our results demonstrate, for the first time, and based on novel patch clamp electrophysiology, potential pharmaco-therapeutic interventions in ME/CFS.

Project description:BackgroundChronic Fatigue Syndrome/ Myalgic Encephalomyelitis (CFS/ME) is a complex multifactorial disorder of unknown cause having multi-system manifestations. Although the aetiology of CFS/ME remains elusive, immunological dysfunction and more particularly reduced cytotoxic activity in natural killer (NK) cells is the most consistent laboratory finding. The Transient Receptor Potential (TRP) superfamily of cation channels play a pivotal role in the pathophysiology of immune diseases and are therefore potential therapeutic targets. We have previously identified single nucleotide polymorphisms in TRP genes in peripheral NK cells from CFS/ME patients. We have also described biochemical pathway changes and calcium signaling perturbations in NK cells from CFS/ME patients. Notably, we have previously reported a decrease of TRP cation channel subfamily melastatin member 3 (TRPM3) function in NK cells isolated from CFS/ME patients compared with healthy controls after modulation with pregnenolone sulfate and ononetin using a patch-clamp technique. In the present study, we aim to confirm the previous results describing an impaired TRPM3 activity in a new cohort of CFS/ME patients using a whole cell patch-clamp technique after modulation with reversible TRPM3 agonists, pregnenolone sulfate and nifedipine, and an effective TRPM3 antagonist, ononetin. Indeed, no formal research has commented on using pregnenolone sulfate or nifedipine to treat CFS/ME patients while there is evidence that clinicians prescribe calcium channel blockers to improve different symptoms.MethodsWhole-cell patch-clamp technique was used to measure TRPM3 activity in isolated NK cells from twelve age- and sex-matched healthy controls and CFS/ME patients, after activation with pregnenolone sulfate and nifedipine and inhibition with ononetin.ResultsWe confirmed a significant reduction in amplitude of TRPM3 currents after pregnenolone sulfate stimulation in isolated NK cells from another cohort of CFS/ME patients compared with healthy controls. The pregnenolone sulfate-evoked ionic currents through TRPM3 channels were again significantly modulated by ononetin in isolated NK cells from healthy controls compared with CFS/ME patients. In addition, we used nifedipine, another reversible TRPM3 agonist to support the previous findings and found similar results confirming a significant loss of the TRPM3 channel activity in CFS/ME patients.ConclusionsImpaired TRPM3 activity was validated in NK cells isolated from CFS/ME patients using different pharmacological tools and whole-cell patch-clamp technique as the gold standard for ion channel research. This investigation further helps to establish TRPM3 channels as a prognostic marker and/ or a potential therapeutic target for CFS/ME.