We have previously determined an optimal in vitro methodology to phenotype TRPM2 and CD38 surface expression on human NK cell subsets from HC participants using flow cytometry [44]. This current investigation is the first in vitro study to characterise TRPM2 and CD38 surface expression on peripheral NK cell subsets from ME/CFS patients. This is also the first study to examine the pharmacological effect of 8-Br-ADPR and N6-Bnz-cAMP drug treatments on TRPM2 and CD38 surface expression, as well as NK cell cytotoxicity in ME/CFS patients.
At baseline, TRPM2 surface expression was significantly higher in ME/CFS patients compared with HCs on CD56BrightCD16Dim/− and (Fig. 1a) and CD56DimCD16+ NK cells (Fig. 1b). These findings were also found at dual expression with CD38 on both NK cell subsets (Fig. 1c, d). CD38 surface expression alone was reportedly higher in ME/CFS and HC participants (99%) on both NK cell subsets (Fig. 2a, b). However, when compared with dual expression with TRPM2, CD38 surface expression decreased to 22% (ME/CFS) and 6% (HC) on both subsets (Fig. 1c, d). This difference with co-expression is reflective of CD38’s additional functions, independent of TRPM2, such as cell adhesion, signal transduction and Ca2+ signalling. However, as CD38 surface expression did not differ between groups, our results highlight an overexpression of the TRPM2 ion channel within the ME/CFS group. In comparison to the reductions in TRPM3 surface expression reported in our previous findings [45, 47], we postulate that this overexpression in TRPM2 may function as a compensatory mechanism to alert a dysregulation in Ca2+ homeostasis within the NK cell.
Ca2+ plays a fundamental role in intracellular signalling pathways, cell differentiation and cell division, apoptosis and transcriptional events [22,23,24]. Upon stimulation, Ca2+ permeable TRP channels, such as TRPM2, generate changes in [Ca2+]i, by acting as Ca2+ gatekeepers via the plasma membrane. Notably, [Ca2+]i is important to sensitise TRPM2 for activation by ADPR resulting in a positive feedback loop and Ca2+ entry [48, 49]. Changes in channel stoichiometry and assembly can induce significant dysregulations in Ca2+ mobilization [50, 51]. Thus, Ca2+ homeostasis requires a tight and meticulous regulation for efficient receptor functionality. An important Ca2+-dependent mechanism regulated by TRPM2 in NK cells is cytotoxic function.
Although the underlying aetiology of ME/CFS remains unknown; a significant reduction in NK cell cytotoxicity is a consistent laboratory finding in ME/CFS patients compared with HCs [3,4,5,6], which was confirmed in this present study (Fig. 3). In the ME/CFS group, an improvement in NK cell function was expected in correlation to the overexpression of TRPM2 as a compensatory mechanism (Fig. 1). However, as baseline NKcell cytotoxicity was significantly reduced in ME/CFS patients compared with HCs, these results may suggest an impaired and/or faulty TRPM2 ion channel within the ME/CFS group. An impairment in the TRPM2 ion channel function may prevent the permeabilization and influx of Ca2+ within the NK cell; resulting in a subsequent reduction in Ca2+ modulation and [Ca2+]i, thus leading to impaired Ca2+-dependent mechanisms, including NK cell cytotoxicity.
Following IL-2 stimulation, NK cell cytotoxicity significantly decreased within the HC group (Fig. 3). This was an unexpected outcome as previous investigations have reported enhancements in NK cell cytotoxicity following IL-2 stimulation [10,11,12]. However, an in vivo mouse study discovered that IL-2 rapidly lowers the activation threshold of NK cells to adhere and engage with their targets [12]. Moreover, NK cell responses did not augment post IL-2 in the presence of inhibitory receptor signalling, suggesting potential interactions of the IL-2R with integrin or activating-receptor signalling pathways [12]. An additional rationale could reflect the limited culture time period (24 h), as most NK cell cultures range between 1 and 2 weeks [52,53,54,55,56]. However, as fresh human peripheral NK cells were used, a longer culture time period was not preferential as NK cell purity significantly decreases to 40–50% [57]. Conversely, no change in NK cell cytotoxicity was observed post IL-2 stimulation within the ME/CFS group (Fig. 3), which reinforces a significant impairment in the TRPM2 ion channel.
Interestingly, TRPM2 and CD38 surface expression significantly decreased after IL-2 stimulation in ME/CFS patients (Fig. 1). However, this finding was only observed on the CD56DimCD16+ NK cell subset within the ME/CFS group (Fig. 1a, c). A possible rationale may involve downregulation the IL-2Rα on the CD56DimCD16+ subset, which has been suggested to reduce the ability for efficient CD56DimCD16+ NK cell activation and restoration of proliferative capability in response to IL-2 [10].
Thus, a negative feedback mechanism, involving Ca2+, may be present between the IL-2Rα and TRPM2 and CD38 on CD56DimCD16+ NK cells in response to IL-2 as it has been proposed that IL-2 may induce the de novo expression of proteins that act between CD38 and the lytic machinery in NK cells [10]. The MAPK signalling pathway may be a potential candidate, as this pathway is activated by both IL-2 and major histocompatibility complex-1 receptor [16, 58], and may mimic mediation of the Ca2+-dependent steps of NK cytotoxicity. Importantly, we have previously shown a significant change in the MAPK Ca2+ dependent pathways for NK lysis in ME/CFS patients [45, 46].
Pharmacologically, TRPM2 currents can be inhibited by altering the production of TRPM2 secondary messengers, such as ADPR [24]. A former in vivo investigation evidenced 8-Br-ADPR as the sole secondary messenger to antagonise TRPM2 by blocking sustained tumour-induced Ca2+ signals and degranulation by western blot and confocal microscopy [28].
Within our ME/CFS group, TRPM2 surface expression significantly decreased following 8-Br-ADPR treatment (ADPR antagonist) on CD56BrightCD16Dim/− NK cells (Fig. 4a). Therefore, TRPM2 surface expression can be antagonised, but not effectively stimulated following agonists, such as N6-Bnz-cAMP (Fig. 4). Interestingly, this result differed with our previous TRPM3 surface expression findings following drug treatment from ME/CFS patients [45, 47]. Following 2‐aminoethoxydiphenyl borate treatment (non-selective TRPM inhibitor), TRPM3 surface expression remained unchanged on both NK cell subsets. Conversely, pregnenolone sulfate (TRPM3 agonist) treatment significantly increased TRPM3 surface expression on both NK cell subsets. Together, these findings reinforce the importance of Ca2+ for efficient TRP channel activity and highlight a consistent Ca2+ signalling dysregulation caused by impairments in TRPM ion channel expression and activity in ME/CFS patients. Moreover, as 8-Br-ADPR was administrated with IL-2, the selective diminishment of the TRPM2 ion channel may be due to the upregulation of the IL-2Rα on the CD56BrightCD16Dim/− subset.
Comparatively, no changes in NK cell cytotoxicity were observed in either group following 8-Br-ADPR and N6-Bnz-cAMP treatment (Fig. 3). These results may reflect the limited availability of specific pharmacological drugs to modulate TRPM2 ion channels. An extensive list of pharmacological agents have been reviewed in the literature including: 8-Br-cADPR (cADPR antagonist) [29], 8-Br-ADPR (ADPR antagonist) [30], N6-Bnz-cAMP (indirect CD38 agonist), nifedipine, econazole [31], flufenamic acid [31], imidazole antifungal agents [31], anthranilic acid, N-(p-amycinnamoyl) [32] and 2-APB [32]. Unfortunately, majority of these molecules are insufficiently potent and do not exhibit high TRPM2 specificity [24], which may explain the absent changes in NK cell cytotoxicity following drug treatment in both groups (Fig. 2).
An alternative rationale may involve different TRPM2 splice variants in ME/CFS patients. Within the literature, three primary TRPM2 isoforms have been established: SSF-TRPM2, TRPM2-ΔC and TRPM2-ΔN [41, 59]. Although the full-length TRPM2 can be activated by ADPR, NAD+ and H2O2, TRPM2 spliced isoforms cannot be significantly stimulated by these same activators [60,61,62]. Interestingly, Du et al. established that 10 µM [Ca2+]i can activate TRPM2-N, TRPM2-C, and TRPM2-N/C spliced isoforms in a concentration-dependent manner. These results suggest that [Ca2+]i may serve as an alternative in vivo activator of both full and spliced isoforms of TRPM2, thus conferring their physiological functions [63]. Whether spliced isoforms can form functional channels remains to be determined [60,61,62].
Our results are considered preliminary due to our small sample size. Resultantly, our significant findings warrant further investigation with a larger cohort as a key future direction. Importantly, a significant limitation within ME/CFS research is the absence of an in vivo model and/or cell line to represent this multifactorial disorder. Consequently, current in vitro methodologies are restricted to studying isolated cells from criteria-based ME/CFS patients. One method to target specific immunological pathways from patient cells is through pharmacological modulation. However, if the mechanism of action of these drugs is unknown or non-specific, severe limitations arise in the ability to analyse accurate and reliable data. Therefore, a vital future direction is the development of pharmacotherapeutic drugs with high efficacy and specificity to TRPM2. Access to these agents will enable the use of more sophisticated applications such as whole cell electrophysiology using patch clamp techniques. Genetic methodologies are an additional future direction to understand the role of TRPM2 in Ca2+ signalling and NK cell function.