Next Article in Journal
Patient-Reported Questionnaires to Identify Adverse Drug Reactions: A Systematic Review
Previous Article in Journal
Predictors of Problematic Social Media Use in a Nationally Representative Sample of Adolescents in Luxembourg
Previous Article in Special Issue
Network Analysis of Symptoms Co-Occurrence in Chronic Fatigue Syndrome
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJERPH
Volume 18
Issue 22
10.3390/ijerph182211879
ijerph-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Characterization of IL-2 Stimulation and TRPM7 Pharmacomodulation in NK Cell Cytotoxicity and Channel Co-Localization with PIP2 in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Patients

by
Stanley Du Preez
1,2,3,4,*,
Natalie Eaton-Fitch
1,2,3,
Helene Cabanas
2,5,
Donald Staines
1,2 and
Sonya Marshall-Gradisnik
1,2
1
National Centre for Neuroimmunology and Emerging Diseases, Menzies Health Institute, Griffith University, Gold Coast 4215, Australia
2
Consortium Health International for Myalgic Encephalomyelitis, Menzies Health Institute Queensland, Griffith University, Gold Coast 4215, Australia
3
School of Pharmacy and Medical Sciences, Griffith University, Gold Coast 4215, Australia
4
School of Medicine and Dentistry, Griffith University, Gold Coast 4215, Australia
5
INSERM U944 and CNRS UMR 7212, Institut de Recherche Saint Louis, Hôpital Saint Louis, APHP, Université de Paris, 75010 Paris, France
*
Author to whom correspondence should be addressed.
Int. J. Environ. Res. Public Health 2021, 18(22), 11879; https://doi.org/10.3390/ijerph182211879
Submission received: 6 October 2021 / Revised: 30 October 2021 / Accepted: 8 November 2021 / Published: 12 November 2021
(This article belongs to the Special Issue Chronic Fatigue Syndrome: Medical, Nursing and Public Health Management)
Browse Figures
Review Reports Versions Notes

Abstract

:
Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex multisystemic disorder responsible for significant disability. Although a unifying etiology for ME/CFS is uncertain, impaired natural killer (NK) cell cytotoxicity represents a consistent and measurable feature of this disorder. Research utilizing patient-derived NK cells has implicated dysregulated calcium (Ca2+) signaling, dysfunction of the phosphatidylinositol-4,5-bisphosphate (PIP2)-dependent cation channel, transient receptor potential melastatin (TRPM) 3, as well as altered surface expression patterns of TRPM3 and TRPM2 in the pathophysiology of ME/CFS. TRPM7 is a related channel that is modulated by PIP2 and participates in Ca2+ signaling. Though TRPM7 is expressed on NK cells, the role of TRPM7 with IL-2 and intracellular signaling mechanisms in the NK cells of ME/CFS patients is unknown. This study examined the effect of IL-2 stimulation and TRPM7 pharmacomodulation on NK cell cytotoxicity using flow cytometric assays as well as co-localization of TRPM7 with PIP2 and cortical actin using confocal microscopy in 17 ME/CFS patients and 17 age- and sex-matched healthy controls. The outcomes of this investigation are preliminary and indicate that crosstalk between IL-2 and TRMP7 exists. A larger sample size to confirm these findings and characterization of TRPM7 in ME/CFS using other experimental modalities are warranted.

1. Introduction

Natural killer (NK) cells are granular lymphocytes that provide anti-viral and anti-tumor defense primarily as part of the innate immune response. The two major human NK cell subsets include cluster of differentiation (CD)56brightCD16dim/- and CD56dimCD16+ NK cells [1]. Immunosurveillance and elimination of transformed autologous cells through cytotoxic effector mechanisms are principally performed by CD56dimCD16+ NK cells circulating in peripheral blood [2,3,4]. NK cell cytotoxicity is mediated by the degranulation of cytotoxic effector molecules including perforin, granzyme, and death ligands stored within secretory lysosomes [5,6,7,8,9]. Importantly, interleukin-2 (IL-2) stimulation significantly enhances the reactivity of NK cells with target cells via a mechanism termed lymphokine-activated killing (LAK) [10,11,12,13].
IL-2 binds the IL-2 receptor (IL-2R) expressed by NK cells and a variety of other immune cells. CD56dimCD16+ NK cells specifically express the intermediate affinity IL-2Rβ, which associates with the common gamma chain subunit forming a heterodimeric signalling complex [10,14]. Downstream mediators of IL-2R activation enhance the transcription of proteins, including cytotoxic effector molecules, that enhance the killing capacity of NK cells [10,13,15]. Once a suitable target cell is identified, an immune synapse (IS) is formed, and the concerted polarization and degranulation of secretory lysosomes within NK cells is regulated by calcium (Ca2+) [16].
Ca2+ is an essential cellular signaling molecule that supports several processes including cell differentiation, activation, apoptosis, and NK cell degranulation [17]. Degranulation processes are activated by increased intracellular Ca2+ following the release of intracellular Ca2+ stores and influx through Ca2+-permeable membrane ion channels. Ca2+ enhances transcriptional events requisite to NK cell cytotoxicity and mediates the translocation of secretory lysosomes and their fusion with the central portion of the IS, resulting in the release of cytotoxic effector molecules into the immune synaptic cleft [16,18,19,20]. Additionally, Ca2+ facilitates the endocytosis of perforin and granzymes and the initiation of apoptotic events in the target cell [18,21,22]. Intracellular Ca2+ and kinase signaling downstream of IS formation and IL-2R ligation are thus essential processes for NK cell cytotoxicity. Importantly, phosphatidylinositol-4,5-bisphosphate (PIP2) serves as a substrate for phosphoinositide 3-kinase (PI3K) in the IL-2 signaling cascade and phospholipase C (PLC) to liberate Ca2+ stores for secretory lysosome translocation and degranulation [23,24,25,26]. Additional crucial proteins that participate in Ca2+ signaling events and PIP2 interactions include the transient receptor potential (TRP) cation channels.
TRP cation channels are polymodal sensors that detect and mediate cellular responses to physical or chemical perturbations in the extracellular milieu particularly through Ca2+ influx [27]. The Ca2+- and magnesium (Mg2+)-permeable cation channel, TRP melastatin member 7 (TRPM7), is constitutively active and depends on the presence of PIP2 to maintain its activity [28,29,30,31]. In turn, TRPM7 modulates the activity of PI3K and PLC according to the availability of intracellular Mg2+ [32,33]. TRPM7 additionally possesses an intrinsic kinase domain that modulates the mitogen-activated protein kinase (MAPK) and PI3K-protein kinase B (Akt)-mammalian target of rapamycin (mTOR) pathways common to IL-2R activation, as well as intracellular Ca2+ signaling mechanisms that are activated as part of NK cell degranulation events [32,33,34,35]. Hence, the TRPM7 cation channel and kinase domains may influence many key signaling events that involve PIP2 and are essential for NK cell activation and effector functions. The role of TRPM7 in NK cell cytotoxicity and interactions with PIP2 can be explored pharmacologically using the TRPM7 agonist, naltriben (NTB), and TRPM7 antagonist, NS8593.
NTB is a specific activator of TRPM7 ionic currents with a reported half-maximal effective concentration (EC50) of 18.1 µM to 24 µM for in vitro models characterizing the effect of immediate TRPM7 stimulation [36]. By contrast, NS8593 blocks the function of both the TRPM7 ion channel and kinase domains with a reported half-maximal inhibitory concentration (IC50) of 5.9 ± 0.6 µM applied in the presence of 300 µM Mg2+ in an in vitro model [37]. A comprehensive review performed by Chubanov and Gudermann summarizes that NS8593 treatment in various cell types replicates the phenotypic features of genetic inactivation of TRPM7 and enables characterization of this channel in human disease [38]. The effect of NS8593 in NK cells, however, was not reported in this review. NTB and NS8593 are therefore appropriate drugs to modulate the activity of TRPM7 to characterize the implications of this protein in the IL-2 priming and cytotoxic mechanisms of NK cells. Importantly, dysregulation of Ca2+ signaling and intracellular mechanisms downstream of IL-2R activation that potentially engage TRPM7 may compromise NK cell function. In clinical research, NK cell dysfunction is a consistent finding in several neuroimmune disorders including myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS).
The pathognomonic features of ME/CFS include fatigue that does not improve with rest, neuroimmune exhaustion, and exacerbation of concomitant symptoms including pain and cognitive impairment after physical or mental exertion [39]. NK cell dysfunction, Ca2+ and kinase signaling impairments have been identified in ME/CFS patients [40,41,42,43]. More recently, TRP ion channel aberrations were implicated in the pathomechanism of ME/CFS. The primary findings generated from in vitro models include reduced TRPM3 surface expression and ion channel function, as well as increased TRPM2 surface expression in the NK cells of ME/CFS patients [41,42,44,45,46]. The phylogenetic and biochemical similarities of TRPM7 with TRPM3 and TRPM2 [27], as well as the potential involvement of TRPM7 in cellular signaling mechanisms that are essential to NK cell function, provide a rationale to characterize TRPM7 in the NK cells of ME/CFS patients.
The implication of TRPM7 in NK cell cytotoxicity and the IL-2 priming mechanism in ME/CFS has not been described. Moreover, the efficacy of the IL-2 cytotoxic priming mechanism in the NK cells of ME/CFS was only explored recently, with data suggesting that IL-2 priming also enhances NK cell cytotoxicity in ME/CFS in vitro [47]. This pilot study, therefore, aims to corroborate the effect of IL-2 stimulation in the NK cells of ME/CFS patients. Secondly, this study is the first in vitro investigation to evaluate the potential role of TRPM7 in NK cell cytotoxicity and the co-localization of TRPM7 with PIP2 after IL-2 stimulation and pharmacological treatment with NTB or NS8593 in the NK cells of both ME/CFS patients and healthy controls (HCs). The significance of these investigations is to help elucidate the pathomechanism of ME/CFS and identify reliable biomarkers for the diagnosis and treatment of this debilitating disorder.

2. Materials and Methods

2.1. Study Participants

A total of 17 ME/CFS and 17 HC participants were included in this pilot study. One participant and the matched control were excluded for abnormal full blood count (FBC) results. Participants were identified through the National Center for Neuroimmunology and Emerging Diseases (NCNED) database and ME/CFS support networks between December 2019 and December 2020. Eligible participants were between 18 and 65 years of age and had a body mass index (BMI) between 18.5 kg/m2 and 30 kg/m2. All ME/CFS patients were classified according to the Canadian Consensus Criteria and the International Consensus Criteria, and age- and sex-matched to healthy controls. Exclusionary criteria were applied to all potential participants and included chronic illness (including autoimmune, cardiac, endocrine, and primary psychiatric disorders), a recent history of smoking, alcohol misuse, pregnancy, or breastfeeding, or administering pharmacological agents that are known to directly or indirectly interfered with TRPM7 channel-kinase activity, Ca2+ signaling, or immune cell activity.
All participants provided written informed consent to participate in the present study approved by the Griffith University Human Research Ethics Committee (HREC/2019/QGC/56469). Eligible participants donated 85 mL of whole venous blood collected in ethylenediaminetetraacetic acid (EDTA) tubes between 7:00 am and 11:00 am in the South-East Queensland, Northern New South Wales, and Melbourne, Victoria regions of Australia. Upon receipt of blood samples to the laboratory, each participant sample was de-identified using a random number and letter generator by an independent blood collector. An FBC was performed within 4 h of collection for each participant as a routine measure for exclusionary hematological abnormalities including features of anemia or white cell derangements.
All participants completed an online questionnaire collecting sociodemographic data, medical history, medications, and symptom history for ME/CFS patients. ME/CFS symptom survey responses were grouped into 10 symptom categories including: (i) cognitive difficulties (slowed thought, impaired concentration, memory consolidation issues); (ii) pain (headaches, muscle pain, multi-joint pain); (iii) sleep disturbances (reversed sleep cycle, disturbed sleep cycle, unrefreshing sleep); (iv) sensory disturbances (sensitivity to touch, vibration, taste, odor, sound; poor coordination or balance); (v) immune disturbances (flu-like symptoms, sore throat, tender lymph nodes); (vi) gastrointestinal disturbances (abdominal pain, nausea, bloating); (vii) cardiovascular disturbances (orthostatic intolerances, lightheadedness, heart palpitations); (viii) respiratory disturbances (difficulty breathing, air hunger); (ix) thermostatic instability (abnormal sweating episodes, hot flushes, cold extremities); and (x) urinary disturbances (changed urination frequency and painful bladder). In addition, a 36-item short-form health survey (SF-36) and World Health Organization (WHO) Disability Assessment Schedule (DAS) were used to determine the level of disability and quality of life.

2.2. Peripheral Blood Mononuclear Cell Isolation and Natural Killer Cell Purification

Eighty mL of whole blood was layered over a density gradient medium (Ficoll-Paque Premium; GE Healthcare, Uppsala, Sweden) and peripheral blood mononuclear cells (PBMCs) were isolated by centrifugation as previously described [48]. The total cell count and viability of PBMCs were determined using trypan blue stain (Bio-rad, Hercules, CA, USA) and adjusted to a final concentration of 5 × 107 cells/mL. An EasySep Negative Human NK Cell Isolation Kit (Stemcell Technologies, Vancouver, BC, Canada) was used to separate NK cells from the PBMCs. Isolated NK cells were stained with CD3-APC-H7 (0.5 µg/5 µL) and CD56-Pe-Cy7 (0.25 µg/5 µL) antibodies (BD Bioscience, Miami, FL, USA) for 30 min at room temperature (RT) protected from light and analyzed using the BD Accuri C6 flow cytometer (BD Bioscience, Miami, FL, USA) to assess purity as previously described [47]. Lymphocyte populations were defined using forward scatter and side scatter measures on dot plots generated by the flow cytometer. The NK cell population was defined as CD3- and CD56+ as previously described [47]. A purity value of ≥90% for NK cell isolates was deemed acceptable (Appendix A, Figure A1).

2.3. Interleukin-2 Stimulation and Drug Treatment

Freshly isolated NK cells were stimulated with 20 IU/mL of recombinant human IL-2 (specific activity 5 × 106 IU/mg) per test (Miltenyi Biotech, BG, Germany). NK cells were additionally treated with NTB (25 µM as determined by dose-response curve, Appendix B, Figure A2) or NTB with NS8593 (10 µM as determined by dose-response curve, Appendix C, Figure A3) (Tocris Bioscience, Sussex, UK) and incubated for 24 h at 37 °C with 5% CO2 in Roswell Park Memorial Institute (RPMI)-1640 medium (Invitrogen Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen Life Technologies, Carlsbad, CA, USA). IL-2-stimulated and drug-treated NK cells were washed with RPMI-1640 supplemented with 10% FBS and prepared for NK cell cytotoxic activity (NKCCA) assays or with 1X phosphate-buffered saline (PBS) pH 7.2 (Invitrogen Life Technologies, Carlsbad, CA, USA) for confocal microscopy.

2.4. Natural Killer Cell Cytotoxic Activity Assay

NKCCA was assessed at baseline and post-IL-2 stimulation/drug treatment as previously described [47]. NK cells were stained with Paul Karl Horan (PKH)-26 (3.5 µL/test) (Sigma-Aldrich, St. Louis, MO, USA) for 5 min and incubated with human erythromyeloblastoid leukemia (K562) target cells for 4 h at 37 °C with 5% CO2 in RPMI-1640 supplemented with 10% FBS. The concentration of NK cells and K562 cells were adjusted to 5 × 105 cells/mL and 1 × 106 cells/mL, respectively, and combined at effector to target ratios (E:T) of 25:1, 12.5:1, and 6.25:1 alongside control conditions for incubation. Control and treated cells were labeled with Annexin V (2.5 µL/test) and 7-AAD (2.5 µL/test) (BD Bioscience, Miami, FL, USA) to assess NKCCA using flow cytometric analysis recording 10,000–30,000 events. NKCCA was calculated as previously described [46] for each E:T as the percent of specific death of K562 cells according to the formula below:
Cytotoxicity   ( % ) = ( early   stage   apoptosis + late   stage   apoptosis + dead   K 562   cells ) All   K 562   cells × 100

2.5. Co-Localization of Transient Receptor Potential Melastatin 7 with Actin and Phosphoinositol 4,5-Bisphosphate in Natural Killer Cells

Confocal microscopy was employed to determine the co-localization of TRPM7 with actin and PIP2 post-IL-2 stimulation/drug treatment. All washes and incubations were performed in 1X PBS pH 7.2 at RT and protected from light unless otherwise stated. NK cells were adhered to glass coverslips using Cell-Tak® (BD Bioscience, East Rutherford, NJ, USA) at 2.5 × 105 cells/coverslip. Freshly isolated NK cells were then fixed with 3% paraformaldehyde (Sigma-Aldrich, St Louis, MO, USA) and permeabilized using 0.2% Tween20 (Sigma-Aldrich, St Louis, MO, USA). Blocking was performed using 3% bovine serum albumin (BSA) in 1X PBS for 1 h. NK cells were incubated with primary antibodies against TRPM7 (0.5 µg/mL) (Abcam, Shanghai, China) and PIP2 (0.3 µg/mL) (Abcam, Shanghai, China) in 3% BSA overnight at 4 °C.
Cells were incubated with Alexa Fluor® 488 secondary antibody (1 µg/mL) (Life Technologies, Carlsbad, CA, USA) against the TRPM7 primary and Alexa Fluor® 594 secondary antibody (1 µg/mL) (Life Technologies, Carlsbad, CA, USA) against the PIP2 primary for 30 min each. Alexa Fluor® 647 (1:500) (Invitrogen, Carlsbad, CA, USA) was applied for 30 min to stain actin, and 4′,6-diamidino-2-phenylindole (300 nM) (Invitrogen, Carlsbad, CA, USA) was applied for 5 min to stain nuclear material. Coverslips were rinsed in deionized water before mounting on glass slides using Mowiol® 4-88 (Sigma-Aldrich, St Louis, MO, USA) mounting media and allowed to dry at 4 °C overnight. PIP2 immunofluorescence was confirmed in non-permeabilized NK cells and TRPM7 immunofluorescence was confirmed using human TRPM7 blocking peptide (Abcam, Shanghai, China). Images were acquired using a Nikon A1 laser scanning confocal microscope (Nikon, Tokyo, Japan) under 60× magnification (Appendixe D and Appendixe E, Figure A4 and Figure A5). Pearson’s and Mander’s correlation coefficients as well as K1 and K2 values were obtained for approximately 8 cells per condition per patient using NIS-Elements image analysis software (Nikon, Tokyo, Japan).

2.6. Statistical Analysis

Pilot data from this investigation were analyzed using GraphPad Prism version 8.0 (GraphPad Software Inc., Version 8, La Jolla, CA, USA). Data distribution and normality were assessed using the Shapiro-Wilk normality test, skewness, and kurtosis. Statistical significance between and within groups for NKCCA and confocal co-localization was assessed using the independent Mann-Whitney U test. Significance was set at p < 0.05 and all data are presented as the mean ± standard error of the mean (SEM) unless otherwise stated.

3. Results

3.1. Participant Demographics

A total of 34 participants (17 HCs and 17 ME/CFS patients) were included in the present study. No significant differences in age, gender, and BMI were identified between groups (Table 1). ME/CFS patients reported greater impairment in all SF-36 items and the WHODAS items, except participation in school/work, compared with HCs. All included participants were within the reference ranges for each FBC parameter and there were no significant differences between groups for these parameters.

3.2. Baseline Natural Killer Cell Cytotoxic Activity

The baseline NKCCA at the E:T of 25:1 for HCs was 28.94 ± 3.893 and 17.13 ± 3.721 for ME/CFS patients, respectively (p < 0.05); at 12.5:1 for HCs was 11.65 ± 1.821 and 7.19 ± 2.120 for ME/CFS patients, respectively (p < 0.05); and at 6.25:1 for HCs was 7.0 ± 1.260 and 2.524 ± 1.182 for ME/CFS patients, respectively (p < 0.001) (Figure 1). At baseline, NK cell cytotoxicity was significantly reduced in the ME/CFS cohort compared with the HC cohort for all E:Ts (25:1, 12.5:1, 6.25:1).

3.3. Natural Killer Cell Cytotoxic Activity Post-Interleukin-2 Stimulation and Drug Treatment

The NKCCA was significantly increased after IL-2 (20 IU/mL) stimulation with the E:T of 25:1 for HCs being 49.67 ± 6.273 and 48.55 ± 5.129 for ME/CFS patients, respectively (p > 0.05); at 12.5:1 for HCs being 49.42 ± 7.256 and 52.68 ± 5.998 for ME/CFS patients, respectively (p > 0.05); and at 6.25:1 for HCs being 39.15 ± 6.347 and 39.08 ± 5.034 for ME/CFS patients, respectively (p > 0.05). Significant differences between baseline lysis and the IL-2 stimulated lysis results within groups were identified (Figure 2). No significant difference was identified between the IL-2 control and the TRPM7 agonism (20IU/mL IL-2 + 25 µM NTB) or TRPM7 antagonism (20 IU/mL IL-2 + 25 µM NTB + 10 µM NS8593) conditions between or within groups for any of the different E:Ts (Figure 3).

3.4. Co-Localization of Transient Receptor Potential Melastatin 7 with Actin

No significant differences were observed for the co-localization of TRPM7 with actin following IL-2 (20 IU/mL) stimulation and treatment with the TRPM7 agonist (20 IU/mL IL-2 + 25 µM NTB) within or between groups. A significant difference between the TRPM7 agonist (20 IU/mL IL-2 + 25 µM NTB) and TRPM7 antagonist (20 IU/mL IL-2 + 25 µM NTB + 10 µM NS8593) treatments was observed within the ME/CFS cohort (p < 0.001) specifically showing an increase in TRPM7 co-localization with actin with TRPM7 antagonism. Additionally, the ME/CFS cohort showed a significant increase in TRPM7 co-localization with actin for the TRPM7 antagonism treatment compared with HCs (p < 0.01) (Figure 4).

3.5. Co-Localization of Transient Receptor Potential Melastatin 7 with Phosphatidylinositol 4,5-Bisphosphate

No significant differences were observed for the co-localization of TRPM7 with PIP2 following IL-2 (20 IU/mL) stimulation and treatment with the TRPM7 agonist (20 IU/mL IL-2 + 25 µM NTB) within or between groups. A significant difference between the TRPM7 agonist (20 IU/mL IL-2 + 25 µM NTB) and TRPM7 antagonist (20 IU/mL IL-2 + 25 µM NTB + 10 µM NS8593) treatments was observed within the HC cohort (p < 0.01) and ME/CFS cohort (p < 0.05) specifically showing an increase in TRPM7 co-localization with PIP2 with TRPM7 antagonism (Figure 5).

4. Discussion

This pilot study supports previous investigations demonstrating significant reductions in baseline NKCCA for ME/CFS patients compared with HCs [49,50,51,52,53,54]. Moreover, this study confirms a previous investigation of significantly enhanced NKCCA following overnight IL-2 stimulation compared with baseline NKCCA in both ME/CFS patients and HCs [47]. This study is novel as it reports a significantly higher co-localization of TRPM7 with actin in the ME/CFS cohort following NS8593 treatment compared with the NTB condition for ME/CFS and the NS8593 condition for HC. For the first time, this study also reports a significant increase in co-localization of TRPM7 with PIP2 in the HC and ME/CFS cohorts following NS8593 treatment compared with the HC NTB and ME/CFS NTB conditions, respectively. The present investigation proposes that crosstalk between intracellular signaling pathways common to IL-2 and TRPM7 may exist.
Importantly, overnight IL-2 incubation of NK cells significantly increased NKCCA in both ME/CFS patients and HCs. This suggests that dysregulation of the IL-2 signaling pathways may not explain the significant reduction in NKCCA that is a consistent feature in ME/CFS patients [49]. IL-2R activation promotes dimerization of the cytoplasmic receptor domains and activation of Janus kinase 1 (JAK1) and JAK3. Signal transducer and activator of transcription 5 (STAT5) is subsequently phosphorylated, which promotes dimerization and translocation of STAT5 to the nucleus. JAK1 additionally activates the MAPK pathway and JAK3 activates the PI3K-Akt-mTOR pathway.
This intracellular signaling cascade is common to TRPM7 kinase signaling and therefore suggests that signaling pathways downstream of IL-2R ligation are shared by TRPM7 and IL-2 [55,56]. Previous investigations have reported impaired Ca2+ storage [41] and intracellular signaling [43] in the NK cell function of ME/CFS patients compared with HCs. The findings of this study suggest that Ca2+ entry through TRPM7 and IL-2 binding to its cognate receptor may provide a crosstalk-dependent pathway for NK cell function. Recently, Eaton-Fitch et al. [47] reported TRPM3 and PIP2 dependent pathway interactions in ME/CFS patients, with a corresponding increase in NKCCA in both ME/CFS and HC following overnight incubation with IL-2.
This study is the first to report that TRPM7 is expressed by NK cells using immunofluorescence and confocal microscopy techniques. Additionally, this study reports the novel finding of TRPM7 co-localizing with actin and PIP2 following NS8593 treatment in ME/CFS patients and HCs. This may likely be due to inhibition of the TRPM7 ion channel and kinase domains necessitating increased co-localization of TRPM7 with PIP2, possibly in an attempt by the cell to maintain TRPM7 activity. Activated PLC hydrolyzes and thereby depletes PIP2 by catalyzing the formation of inositol triphosphate and diacylglycerol, which promote intracellular Ca2+ store release and MAPK signaling cascades, respectively [57]. PLC and MAPK signaling networks regulate cellular mechanisms that recruit and activate other signaling proteins, reorganize the cytoskeleton, mobilize Ca2+, and activate NK cell effector functions [58,59,60,61].
It is known that the availability of PIP2 influences the activity of TRPM7 according to a biphasic model. Transient depletion of PIP2 from the activation of PLC potentiates TRPM7 ionic currents, and exhaustion of PIP2 reserves beyond an unknown limit renders TRPM7 inactive [62,63,64,65,66,67,68]. In turn, TRPM7 modulates PLC activity and Ca2+ signaling. Under conditions of low intracellular Mg2+ levels, the TRPM7 kinase phosphorylates PLC, thereby downregulating Ca2+ signaling [32]. Hence, significantly increased co-localization of TRPM7 with PIP2 following inhibition of the channel and kinase domains may indicate a mechanism to re-establish TRPM7 activity, which is speculated to be more effective in HCs compared with ME/CFS patients due to dysregulation of Ca2+-dependent processes including protein trafficking.
From this investigation, it was also found that TRPM7 co-localization potentially increases to support cell surface signaling mechanisms such as Ca2+ influx, exocytosis, and kinase signaling. Alternatively, this finding may be explained as an artifact from TRPM7 co-localizing with PIP2, which is more abundant at the plasma membrane. Interestingly, Balinas et al., reported a significant increase in TRMP2 as a possible compensatory mechanism for reduced NKCCA in ME/CFS patients compared to HCs [46]; however, PIP2 was not assessed.
This current investigation additionally reported significant changes in co-localization of TRPM7 with cortical actin of NK cells in HCs, as well as ME/CFS patients, to a lesser extent. TRPM7 expression on the surface of cell membranes is normally scarce [69] although comparatively greater in cells derived from the hematopoietic lineage, including lymphocytes, especially when activated [70]. Rather, TRPM7 predominantly exists on the surface of intracellular vesicles [69] and may be mobilized to the surface when this channel needs to be replenished.
Classically, the soluble N-ethylmaleimide-sensitive-factor accessory protein-receptor (SNARE) proteins are key mediators that facilitate vesicle packaging and fusion at the cell membrane and require adequate intracellular Ca2+ concentrations to function [71]. Thus, translocation of TRP ion channels, in this instance TRPM7, may be supported by local increases in Ca2+. Differences in co-localization of TRPM7 in ME/CFS compared with HCs may therefore reflect the known Ca2+ dysregulation in ME/CFS and supported by previous dysfunctional TRPM3 and Ca2+ signaling findings reported in ME/CFS patients [41,42,44,45,46,47,72]. Future investigations are indicated to determine whether compromised Ca2+ mobilization in ME/CFS patients is indeed responsible for impaired protein translocation and expression.
Limitations of this pilot study include the finite number of cells that were able to be isolated from donor blood samples for experimental work. Consequentially, this restricted the scope of outcomes that could be addressed by the present study. Further investigations examining the intracellular signaling pathways associated with TRPM7 may elucidate the findings reported in this current project. Moreover, no pre-existing literature regarding the effect of treatment with NS8593 alone in NK cells was available. While potential off-target effects may exist for NS8593 and NTB, there is a paucity of literature in NK cells. Using the latest literature, NS8593 and NTB were deemed the most appropriate pharmacological tools for characterizing TRPM7 channel activity in the NK cells of ME/CFS patients. Extending this pilot investigation with further research to characterize TRPM7 activity in the NK cells of ME/CFS patients may, therefore, augment the current hypothesis of pathophysiological mechanisms in ME/CFS.

5. Conclusions

The results from this investigation are preliminary and suggest crosstalk between IL-2 and TRMP7 exists. However, a larger sample size to confirm these findings is warranted. Importantly, these current data, along with previous studies reporting TRP dysfunction and altered expression patterns in the NK cells of ME/CFS patients provide evidence for potential channelopathy as the pathomechanism for ME/CFS. Prospective Ca2+ influx assays, isotyping, and patch-clamp electrophysiology provide additional avenues for further characterizing TRPM7 in the pathomechanism of this illness for possible targeted pharmacotherapeutic interventions for the treatment of ME/CFS.

Author Contributions

Conceptualization, H.C. and S.M.-G.; methodology, S.D.P., N.E.-F., H.C., S.M.-G.; formal analysis, S.D.P.; investigation S.D.P., N.E.-F.; writing—original draft preparation, S.D.P.; writing—review and editing, H.C., D.S., S.M.-G.; visualization, S.D.P.; supervision, H.C., D.S., S.M.-G.; project administration, D.S., S.M.-G.; funding acquisition, D.S., S.M.-G. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Stafford Fox Medical Research Foundation (grant number 489798), Ian and Talei Stewart, McCusker Charitable Foundation (grant number 49979), Buxton Foundation (grant number 4676), Henty Community (grant number 4879), Henty Lions Club (grant number 4880), Mason Foundation (grant number 47107), Mr Douglas Stutt, Blake Beckett Trust Foundation (grant number 4579), Alison Hunter Memorial Foundation (grant number 4570) and the Change for ME Charity (grant number 4575).

Institutional Review Board Statement

The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Human Research Ethics Committee of Griffith University (HREC/2019/QGC/56469 approved 30th September 2019).

Informed Consent Statement

Informed consent was obtained from all subjects involved in the study.

Acknowledgments

The authors would like to acknowledge those who volunteered their time for this project. The authors would also like to acknowledge Cameron Flegg for his expertise in confocal microscopy.

Conflicts of Interest

The authors declare no conflict of interest.

Appendix A

NK cell purity after isolation was 91.91 ± 0.9710 for HCs and 90.68 ± 0.9895 for ME/CFS patients with no significant difference between groups (p > 0.05).
Ijerph 18 11879 g0a1 550
Figure A1. NK cell purity; n = 17 for HC and n = 17 for ME/CFS. NK cells were stained with CD3-APC-H7 (0.5 µg/5 µL) and CD56-Pe-Cy7 (0.25 µg/5 µL) and acquired by flow cytometry. NK cells were defined as the CD3-CD56+ lymphocyte population. Data are represented as the mean ± SEM as determined by independent Mann-Whitney U tests.
Figure A1. NK cell purity; n = 17 for HC and n = 17 for ME/CFS. NK cells were stained with CD3-APC-H7 (0.5 µg/5 µL) and CD56-Pe-Cy7 (0.25 µg/5 µL) and acquired by flow cytometry. NK cells were defined as the CD3-CD56+ lymphocyte population. Data are represented as the mean ± SEM as determined by independent Mann-Whitney U tests.
Ijerph 18 11879 g0a1

Appendix B

NK cells isolated from HCs (n = 4) were used to determine the EC50 of NTB through flow cytometric cell lysis assays as described in Methods Section 2.4. NTB (0, 3, 10, 30, 100 µM; range tested according to values reported in the literature) (Sigma-Aldrich, St. Louis, MO, USA) was prepared in RPMI-1640 supplemented with 10% FBS and IL-2 (20 IU/mL) (Miltenyi Biotec, Bergisch Gladbach, Germany). NK cells at a concentration of 5 × 105 cells per condition were treated for 24 h under physiological conditions. NK cytotoxicity was assessed using flow cytometry. The EC50 of NTB was determined to be 27.32 µM (Figure A2).
The literature reported varied EC50 values due to different lengths of exposure, cell types, and methodologies. For example, the patch-clamp experiments require higher NTB concentrations because the exposure is brief. A participant showed reduced lysis at 30 and 100 µM NTB (data not shown). Additionally, NTB at higher concentrations suppresses lymphocyte proliferation by a mechanism not mediated through TRPM7 or the δ-opioid receptor [73,74,75]. Immunosuppression was not demonstrated in this dose response curve. However, to limit off-target effects of NTB, the concentration used in subsequent experiments for Phase I was rounded down to 25 µM instead of up to 30 µM as other patients may also be susceptible to these immunosuppressive effects at higher NTB concentrations.
Ijerph 18 11879 g0a2 550
Figure A2. EC50 of NTB assessed by NKCCA. The EC50 of NTB was determined using flow cytometric cell lysis assays where NK cells isolated from HCs were treated with NTB (0, 3, 10, 30, 100 µM) and IL-2 (20 IU/mL) for 24 h to activate TRPM7 before incubating with K562 (n = 4). Statistical analysis determined the EC50 of NTB under these conditions to be 27.32 µM and is consistent with the literature. Abbreviations: half-maximal effective concentration (EC50), healthy controls (HCs), naltriben (NTB), natural killer cell cytotoxic activity (NKCCA), interleukin-2 (IL-2), transient receptor potential melastatin 7 (TRPM7), myelogenous leukemia target cell line (K562).
Figure A2. EC50 of NTB assessed by NKCCA. The EC50 of NTB was determined using flow cytometric cell lysis assays where NK cells isolated from HCs were treated with NTB (0, 3, 10, 30, 100 µM) and IL-2 (20 IU/mL) for 24 h to activate TRPM7 before incubating with K562 (n = 4). Statistical analysis determined the EC50 of NTB under these conditions to be 27.32 µM and is consistent with the literature. Abbreviations: half-maximal effective concentration (EC50), healthy controls (HCs), naltriben (NTB), natural killer cell cytotoxic activity (NKCCA), interleukin-2 (IL-2), transient receptor potential melastatin 7 (TRPM7), myelogenous leukemia target cell line (K562).
Ijerph 18 11879 g0a2

Appendix C

Similarly, NK cells isolated from HCs were used to determine the IC50 of NS8593 (Sigma-Aldrich, St. Louis, MO, USA) through flow cytometric cell lysis assays as described in Methods Section 2.4. A range of NS8593 concentrations (0, 1, 3, 10, 30 µM; range tested according to values reported in the literature) were prepared in RPMI with 10% FBS and IL-2 (20 IU/mL) with the optimized NTB concentration (25 µM) and used to treat NK cells at a concentration of 5 × 105 cells per condition for 24 h under physiological conditions. NK cytotoxicity was assessed using flow cytometry. The IC50 of NS8593 was determined to be 7.72 µM (Figure A3). Subsequent experiments used 10 µM NS8593 to ensure TRPM7 blockade was achieved.
Ijerph 18 11879 g0a3 550
Figure A3. IC50 of NS8593 assessed by NKCCA. The IC50 of NS8593 was determined using flow cytometric cell lysis assays where NK cells isolated from HCs were treated with NS8593 (0, 3, 10, 30 µM), IL-2 (20 IU/mL), and NTB (25µM) for 24 h to inhibit TRPM7 before incubating with K562 for cell lysis to occur (n = 4). Statistical analysis determined the IC50 of NS8593 under these conditions to be 7.72 µM and this is consistent with values reported in the literature. Abbreviations: healthy controls (HCs), half maximal inhibitory concentration (IC50), natural killer cell cytotoxic activity (NKCCA), naltriben (NTB), interleukin-2 (IL-2), natural killer (NK), transient receptor potential melastatin 7 (TRPM7), myelogenous leukemia target cell line (K562).
Figure A3. IC50 of NS8593 assessed by NKCCA. The IC50 of NS8593 was determined using flow cytometric cell lysis assays where NK cells isolated from HCs were treated with NS8593 (0, 3, 10, 30 µM), IL-2 (20 IU/mL), and NTB (25µM) for 24 h to inhibit TRPM7 before incubating with K562 for cell lysis to occur (n = 4). Statistical analysis determined the IC50 of NS8593 under these conditions to be 7.72 µM and this is consistent with values reported in the literature. Abbreviations: healthy controls (HCs), half maximal inhibitory concentration (IC50), natural killer cell cytotoxic activity (NKCCA), naltriben (NTB), interleukin-2 (IL-2), natural killer (NK), transient receptor potential melastatin 7 (TRPM7), myelogenous leukemia target cell line (K562).
Ijerph 18 11879 g0a3

Appendix D

Ijerph 18 11879 g0a4 550
Figure A4. Representative composite images of NK cells from a HC following pharmacomodulation of TRPM7. Co-localisation of TRPM7 (green) with (A) PIP2 (red) and (B) actin (white) with the nucleus in blue. Individual images of (C) TRPM7, (D) PIP2 (E) actin (F) nucleus also presented. Abbreviations: natural killer (NK), transient receptor potential melastatin 7 (TRPM7), phosphatidylinositol 4,5-bisphosphate (PIP2).
Figure A4. Representative composite images of NK cells from a HC following pharmacomodulation of TRPM7. Co-localisation of TRPM7 (green) with (A) PIP2 (red) and (B) actin (white) with the nucleus in blue. Individual images of (C) TRPM7, (D) PIP2 (E) actin (F) nucleus also presented. Abbreviations: natural killer (NK), transient receptor potential melastatin 7 (TRPM7), phosphatidylinositol 4,5-bisphosphate (PIP2).
Ijerph 18 11879 g0a4

Appendix E

Ijerph 18 11879 g0a5 550
Figure A5. Representative composite images of NK cells from a ME/CFS patient following pharmacomodulation of TRPM7. Co-localisation of TRPM7 (green) with (A) PIP2 (red) and (B) actin (white) with the nucleus in blue. Individual images of (C) TRPM7, (D) PIP2 (E) actin (F) nucleus also presented. Abbreviations: natural killer (NK), transient receptor potential melastatin 7 (TRPM7), phosphatidylinositol 4,5-bisphosphate (PIP2).
Figure A5. Representative composite images of NK cells from a ME/CFS patient following pharmacomodulation of TRPM7. Co-localisation of TRPM7 (green) with (A) PIP2 (red) and (B) actin (white) with the nucleus in blue. Individual images of (C) TRPM7, (D) PIP2 (E) actin (F) nucleus also presented. Abbreviations: natural killer (NK), transient receptor potential melastatin 7 (TRPM7), phosphatidylinositol 4,5-bisphosphate (PIP2).
Ijerph 18 11879 g0a5

References

  1. Vivier, E.; Tomasello, E.; Baratin, M.; Walzer, T.; Ugolini, S. Functions of natural killer cells. Nat. Immunol. 2008, 9, 503–510. [Google Scholar] [CrossRef]
  2. Fehniger, T.; Cooper, M.; Nuovo, G.J.; Cella, M.; Facchetti, F.; Colonna, M.; Caligiuri, M.A. CD56bright natural killer cells are present in human lymph nodes and are activated by T cell-derived IL-2: A potential new link between adaptive and innate immunity. Blood 2003, 101, 3052–3057. [Google Scholar] [CrossRef] [Green Version]
  3. Caligiuri, M.A. Human natural killer cells. Blood 2008, 112, 461–469. [Google Scholar] [CrossRef]
  4. Poli, A.; Michel, T.; Thérésine, M.; Andrès, E.; Hentges, F.; Zimmer, J. CD56brightnatural killer (NK) cells: An important NK cell subset. Immunology 2009, 126, 458–465. [Google Scholar] [CrossRef]
  5. Lopez, J.A.; Brennan, A.J.; Whisstock, J.C.; Voskoboinik, I.; Trapani, J.A. Protecting a serial killer: Pathways for perforin trafficking and self-defence ensure sequential target cell death. Trends Immunol. 2012, 33, 406–412. [Google Scholar] [CrossRef]
  6. Voskoboinik, I.; Whisstock, J.; Trapani, J. Perforin and granzymes: Function, dysfunction and human pathology. Nat. Rev. Immunol. 2015, 15, 388–400. [Google Scholar] [CrossRef]
  7. Bolitho, P.; Voskoboinik, I.; Trapani, J.; Smyth, M.J. Apoptosis induced by the lymphocyte effector molecule perforin. Curr. Opin. Immunol. 2007, 19, 339–347. [Google Scholar] [CrossRef]
  8. Wang, S.; El-Deiry, W.S. TRAIL and apoptosis induction by TNF-family death receptors. Oncogene 2003, 22, 8628–8633. [Google Scholar] [CrossRef] [Green Version]
  9. Screpanti, V.; Wallin, R.P.; Grandien, A.; Ljunggren, H.-G. Impact of FASL-induced apoptosis in the elimination of tumor cells by NK cells. Mol. Immunol. 2005, 42, 495–499. [Google Scholar] [CrossRef]
  10. Olejniczak, K.; Kasprzak, A. Biological properties of interleukin 2 and its role in pathogenesis of selected diseases—A review. Med. Sci. Monit. 2008, 14, RA179–RA189. [Google Scholar]
  11. Henney, C.S.; Kuribayashi, K.; Kern, D.E.; Gillis, S. Interleukin-2 augments natural killer cell activity. Nature 1981, 291, 335–338. [Google Scholar] [CrossRef]
  12. Carson, W.E.; Parihar, R.; Lindemann, M.J.; Personeni, N.; Dierksheide, J.; Meropol, N.J.; Baselga, J.; Caligiuri, M.A. Interleukin-2 enhances the natural killer cell response to Herceptin-coated Her2/neu-positive breast cancer cells. Eur. J. Immunol. 2001, 31, 3016–3025. [Google Scholar] [CrossRef]
  13. Mirandola, P.; Ponti, C.; Gobbi, G.; Sponzilli, I.; Melloni, E.; Vitale, M. The response of human natural killer cells to interleukin-2. J. Endocrinol. Invest. 2004, 27, 146–150. [Google Scholar]
  14. Cooper, M.; Fehniger, T.; Caligiuri, M.A. The biology of human natural killer-cell subsets. Trends Immunol. 2001, 22, 633–640. [Google Scholar] [CrossRef]
  15. Becknell, B.; Caligiuri, M.A. Interleukin-2, Interleukin-15, and Their Roles in Human Natural Killer Cells. Adv. Immunol. 2005, 86, 209–239. [Google Scholar] [CrossRef]
  16. Hartzell, C.A.; Jankowska, K.I.; Burkhardt, J.; Lewis, R.S. Calcium influx through CRAC channels controls actin organization and dynamics at the immune synapse. eLife 2016, 5, e14850. [Google Scholar] [CrossRef]
  17. Clapham, D.E. Calcium Signaling. Cell 2007, 131, 1047–1058. [Google Scholar] [CrossRef] [Green Version]
  18. Krzewski, K.; Coligan, J.E. Human NK cell lytic granules and regulation of their exocytosis. Front. Immunol. 2012, 3, 335. [Google Scholar] [CrossRef] [Green Version]
  19. Masi, G.; Baldari, C.T. Signaling at the immune synapse: Vesicular trafficking takes the stage. Cell. Mol. Immunol. 2013, 10, 459–462. [Google Scholar] [CrossRef] [Green Version]
  20. Smyth, M.J.; Cretney, E.; Kelly, J.M.; Westwood, J.A.; Street, S.E.; Yagita, H.; Takeda, K.; van Dommelen, S.L.; Degli-Esposti, M.A.; Hayakawa, Y. Activation of NK cell cytotoxicity. Mol. Immunol. 2005, 42, 501–510. [Google Scholar] [CrossRef]
  21. Nicotera, P.; Orrenius, S. The role of calcium in apoptosis. Cell Calcium 1998, 23, 173–180. [Google Scholar] [CrossRef]
  22. Pinton, P.; Giorgi, C.; Siviero, R.; Zecchini, E.; Rizzuto, R. Calcium and apoptosis: ER-mitochondria Ca2+ transfer in the control of apoptosis. Oncogene 2008, 27, 6407–6418. [Google Scholar] [CrossRef] [Green Version]
  23. Enandagopal, N.; Ali, A.K.; Komal, A.K.; Elee, S.-H. The Critical Role of IL-15-PI3K-mTOR Pathway in Natural Killer Cell Effector Functions. Front. Immunol. 2014, 5, 187. [Google Scholar] [CrossRef]
  24. Tassi, I.; Klesney-Tait, J.; Colonna, M. Dissecting natural killer cell activation pathways through analysis of genetic mutations in human and mouse. Immunol. Rev. 2006, 214, 92–105. [Google Scholar] [CrossRef]
  25. Broad, L.M.; Braun, F.-J.; Lievremont, J.-P.; Bird, G.S.J.; Kurosaki, T., Jr.; Putney, J.W. Role of the Phospholipase C-Inositol 1,4,5-Trisphosphate Pathway in Calcium Release-activated Calcium Current and Capacitative Calcium Entry. J. Biol. Chem. 2001, 276, 15945–15952. [Google Scholar] [CrossRef] [Green Version]
  26. Putney, J.W.; Steinckwich-Besançon, N.; Numaga-Tomita, T.; Davis, F.M.; Desai, P.N.; D’Agostin, D.M.; Wu, S.; Bird, G.S. The functions of store-operated calcium channels. Biochim. Biophys. Acta Bioenerg. 2016, 1864, 900–906. [Google Scholar] [CrossRef]
  27. Nilius, B.; Owsianik, G. The transient receptor potential family of ion channels. Genome Biol. 2011, 12, 1–11. [Google Scholar] [CrossRef] [Green Version]
  28. Nadler, M.J.S.; Hermosura, M.C.; Inabe, K.; Perraud, A.-L.; Zhu, Q.; Stokes, A.; Kurosaki, T.; Kinet, J.-P.; Penner, R.; Scharenberg, A.M.; et al. LTRPC7 is a Mg·ATP-regulated divalent cation channel required for cell viability. Nat. Cell Biol. 2001, 411, 590–595. [Google Scholar] [CrossRef]
  29. Schmitz, C.; Perraud, A.-L.; Johnson, C.O.; Inabe, K.; Smith, M.K.; Penner, R.; Kurosaki, T.; Fleig, A.; Scharenberg, A.M. Regulation of Vertebrate Cellular Mg2+ Homeostasis by TRPM7. Cell 2003, 114, 191–200. [Google Scholar] [CrossRef] [Green Version]
  30. Monteilh-Zoller, M.K.; Hermosura, M.C.; Nadler, M.J.; Scharenberg, A.M.; Penner, R.; Fleig, A. TRPM7 Provides an Ion Channel Mechanism for Cellular Entry of Trace Metal Ions. J. Gen. Physiol. 2002, 121, 49–60. [Google Scholar] [CrossRef] [Green Version]
  31. Smyth, J.T.; Hwang, S.-Y.; Tomita, T.; DeHaven, W.I.; Mercer, J.C.; Putney, J.W. Activation and regulation of store-operated calcium entry. J. Cell. Mol. Med. 2010, 14, 2337–2349. [Google Scholar] [CrossRef]
  32. Deason-Towne, F.; Perraud, A.-L.; Schmitz, C. Identification of Ser/Thr phosphorylation sites in the C2-domain of phospholipase C γ2 (PLCγ2) using TRPM7-kinase. Cell. Signal. 2012, 24, 2070–2075. [Google Scholar] [CrossRef] [Green Version]
  33. Zou, Z.-G.; Rios, F.J.; Montezano, A.C.; Touyz, R.M. TRPM7, Magnesium, and Signaling. Int. J. Mol. Sci. 2019, 20, 1877. [Google Scholar] [CrossRef] [Green Version]
  34. Faouzi, M.; Kilch, T.; Horgen, F.D.; Fleig, A.; Penner, R. The TRPM7 channel kinase regulates store-operated calcium entry. J. Physiol. 2017, 595, 3165–3180. [Google Scholar] [CrossRef] [Green Version]
  35. Beesetty, P.; Wieczerzak, K.B.; Gibson, J.N.; Kaitsuka, T.; Luu, C.T.; Matsushita, M.; Kozak, J.A. Inactivation of TRPM7 kinase in mice results in enlarged spleens, reduced T-cell proliferation and diminished store-operated calcium entry. Sci. Rep. 2018, 8, 1–22. [Google Scholar] [CrossRef]
  36. Hofmann, T.; Schäfer, S.; Linseisen, M.; Sytik, L.; Gudermann, T.; Chubanov, V. Activation of TRPM7 channels by small molecules under physiological conditions. Eur. J. Physiol. 2014, 466, 2177–2189. [Google Scholar] [CrossRef]
  37. Chubanov, V.; Schäfer, S.; Ferioli, S.; Gudermann, T. Natural and Synthetic Modulators of the TRPM7 Channel. Cells 2014, 3, 1089–1101. [Google Scholar] [CrossRef]
  38. Chubanov, V.; Gudermann, T. Mapping TRPM7 Function by NS8593. Int. J. Mol. Sci. 2020, 21, 7017. [Google Scholar] [CrossRef]
  39. Carruthers, B.M.; van de Sande, M.I.; de Meirleir, K.L.; Klimas, N.G.; Broderick, G.; Mitchell, T.; Staines, D.; Powles, A.C.P.; Speight, N.; Vallings, R.; et al. Myalgic encephalomyelitis: International Consensus Criteria. J. Intern. Med. 2011, 270, 327–338. [Google Scholar] [CrossRef] [Green Version]
  40. Chacko, A.; Staines, D.R.; Johnston, S.C.; Marshall-Gradisnik, S.M. Dysregulation of Protein Kinase Gene Expression in NK Cells from Chronic Fatigue Syndrome/Myalgic Encephalomyelitis Patients. Gene Regul. Syst. Biol. 2016, 10, 85–93. [Google Scholar] [CrossRef]
  41. Nguyen, T.; Staines, D.; Nilius, B.; Smith, P.; Marshall-Gradisnik, S. Novel identification and characterisation of Transient receptor potential melastatin 3 ion channels on Natural Killer cells and B lymphocytes: Effects on cell signalling in Chronic fatigue syndrome/Myalgic encephalomyelitis patients. Biol. Res. 2016, 49, 1–8. [Google Scholar] [CrossRef] [Green Version]
  42. Nguyen, T.; Johnston, S.; Clarke, L.; Smith, P.; Staines, D.; Marshall-Gradisnik, S. Impaired calcium mobilization in natural killer cells from chronic fatigue syndrome/myalgic encephalomyelitis patients is associated with transient receptor potential melastatin 3 ion channels. Clin. Exp. Immunol. 2016, 187, 284–293. [Google Scholar] [CrossRef] [Green Version]
  43. Huth, T.K.; Staines, D.; Marshall-Gradisnik, S. ERK1/2, MEK1/2 and p38 downstream signalling molecules impaired in CD56 dim CD16+ and CD56 bright CD16 dim/- natural killer cells in Chronic Fatigue Syndrome/Myalgic Encephalomyelitis patients. J. Transl. Med. 2016, 14, 97. [Google Scholar] [CrossRef] [Green Version]
  44. Cabanas, H.; Muraki, K.; Eaton, N.; Balinas, C.; Staines, D.; Marshall-Gradisnik, S. Loss of Transient Receptor Potential Melastatin 3 ion channel function in natural killer cells from Chronic Fatigue Syndrome/Myalgic Encephalomyelitis patients. Mol. Med. 2018, 24, 1–10. [Google Scholar] [CrossRef]
  45. Cabanas, H.; Muraki, K.; Balinas, C.; Eaton-Fitch, N.; Staines, D.; Marshall-Gradisnik, S. Validation of impaired Transient Receptor Potential Melastatin 3 ion channel activity in natural killer cells from Chronic Fatigue Syndrome/Myalgic Encephalomyelitis patients. Mol. Med. 2019, 25, 14. [Google Scholar] [CrossRef] [Green Version]
  46. Balinas, C.; Cabanas, H.; Staines, D.; Marshall-Gradisnik, S. Transient receptor potential melastatin 2 channels are overexpressed in myalgic encephalomyelitis/chronic fatigue syndrome patients. J. Transl. Med. 2019, 17, 1–11. [Google Scholar] [CrossRef]
  47. Eaton-Fitch, N.; Cabanas, H.; du Preez, S.; Staines, D.; Marshall-Gradisnik, S. The effect of IL-2 stimulation and treatment of TRPM3 on channel co-localisation with PIP2 and NK cell function in myalgic encephalomyelitis/chronic fatigue syndrome patients. J. Transl. Med. 2021, 19, 1–19. [Google Scholar] [CrossRef]
  48. Panda, S.K.; Ravindran, B. Isolation of Human PBMCs. BioProtoc. 2013, 3, e323. [Google Scholar] [CrossRef]
  49. Eaton-Fitch, N.; Du Preez, S.; Cabanas, H.; Staines, D.; Marshall-Gradisnik, S. A systematic review of natural killer cells profile and cytotoxic function in myalgic encephalomyelitis/chronic fatigue syndrome. Syst. Rev. 2019, 8, 1–13. [Google Scholar] [CrossRef]
  50. Klimas, N.G.; Salvato, F.R.; Morgan, R.; Fletcher, M.A. Immunologic abnormalities in chronic fatigue syndrome. J. Clin. Microbiol. 1990, 28, 1403–1410. [Google Scholar] [CrossRef] [Green Version]
  51. Maher, K.J.; Klimas, N.G.; Fletcher, M.A. Chronic fatigue syndrome is associated with diminished intracellular perforin. Clin. Exp. Immunol. 2005, 142, 505–511. [Google Scholar] [CrossRef]
  52. Fletcher, M.A.; Zeng, X.R.; Maher, K.; Levis, S.; Hurwitz, B.; Antoni, M.; Broderick, G.; Klimas, N.G. Biomarkers in Chronic Fatigue Syndrome: Evaluation of Natural Killer Cell Function and Dipeptidyl Peptidase IV/CD26. PLoS ONE 2010, 5, e10817. [Google Scholar] [CrossRef]
  53. Brenu, E.W.; van Driel, M.L.; Staines, D.R.; Ashton, K.J.; Ramos, S.B.; Keane, J.; Klimas, N.G.; Marshall-Gradisnik, S.M. Immunological abnormalities as potential biomarkers in Chronic Fatigue Syndrome/Myalgic Encephalomyelitis. J. Transl. Med. 2011, 9, 81. [Google Scholar] [CrossRef] [Green Version]
  54. Brenu, E.W.; Van Driel, M.L.; Staines, D.R.; Ashton, K.J.; Hardcastle, S.L.; Keane, J.; Tajouri, L.; Peterson, D.; Ramos, S.B.; Marshall-Gradisnik, S.M. Longitudinal investigation of natural killer cells and cytokines in chronic fatigue syndrome/myalgic encephalomyelitis. J. Transl. Med. 2012, 10, 88. [Google Scholar] [CrossRef] [Green Version]
  55. Fan, M.Y.; Turka, L.A. Immunometabolism and PI(3)K Signaling As a Link between IL-2, Foxp3 Expression, and Suppressor Function in Regulatory T Cells. Front. Immunol. 2018, 9, 69. [Google Scholar] [CrossRef] [Green Version]
  56. Kobayashi, T.; Mattarollo, S.R. Natural killer cell metabolism. Mol. Immunol. 2019, 115, 3–11. [Google Scholar] [CrossRef]
  57. Goldsmith, Z.G.; Dhanasekaran, D.N. G Protein regulation of MAPK networks. Oncogene 2007, 26, 3122–3142. [Google Scholar] [CrossRef] [Green Version]
  58. Chen, X.; Allan, D.S.J.; Krzewski, K.; Ge, B.; Kopcow, H.; Strominger, J.L. CD28-stimulated ERK2 phosphorylation is required for polarization of the microtubule organizing center and granules in YTS NK cells. Proc. Natl. Acad. Sci. USA 2006, 103, 10346–10351. [Google Scholar] [CrossRef] [Green Version]
  59. Chuderland, D.; Seger, R. Calcium regulates ERK signaling by modulating its protein-protein interactions. Commun. Integr. Biol. 2008, 1, 4–5. [Google Scholar] [CrossRef] [Green Version]
  60. Lanier, L.L. Up on the tightrope: Natural killer cell activation and inhibition. Nat. Immunol. 2008, 9, 495–502. [Google Scholar] [CrossRef]
  61. Schwarz, E.C.; Qu, B.; Hoth, M. Calcium, cancer and killing: The role of calcium in killing cancer cells by cytotoxic T lymphocytes and natural killer cells. Biochim. Biophys. Acta Bioenerg. 2013, 1833, 1603–1611. [Google Scholar] [CrossRef] [Green Version]
  62. Clark, K.; Langeslag, M.; Van Leeuwen, B.; Ran, L.; Ryazanov, A.G.; Figdor, C.; Moolenaar, W.H.; Jalink, K.; van Leeuwen, F.N. TRPM7, a novel regulator of actomyosin contractility and cell adhesion. EMBO J. 2006, 25, 290–301. [Google Scholar] [CrossRef]
  63. Runnels, L.; Yue, L.; Clapham, D. The TRPM7 channel is inactivated by PIP2 hydrolysis. Nat. Cell Biol. 2002, 4, 329–336. [Google Scholar] [CrossRef]
  64. Langeslag, M.; Clark, K.; Moolenaar, W.H.; van Leeuwen, F.N.; Jalink, K. Activation of TRPM7 Channels by Phospholipase C-coupled Receptor Agonists. J. Biol. Chem. 2007, 282, 232–239. [Google Scholar] [CrossRef] [Green Version]
  65. Macianskiene, R.; Gwanyanya, A.; Vereecke, J.; Mubagwa, K. Inhibition of the Magnesium-Sensitive TRPM7-like Channel in Cardiac Myocytes by Nonhydrolysable GTP Analogs: Involvement of Phosphoinositide Metabolism. Cell. Physiol. Biochem. 2008, 22, 109–118. [Google Scholar] [CrossRef]
  66. Callera, G.E.; He, Y.; Yogi, A.; Montezano, A.; Paravicini, T.; Yao, G.; Touyz, R.M. Regulation of the novel Mg2+ transporter transient receptor potential melastatin 7 (TRPM7) cation channel by bradykinin in vascular smooth muscle cells. J. Hypertens. 2009, 27, 155–166. [Google Scholar] [CrossRef]
  67. Yogi, A.; Callera, G.E.; Tostes, R.; Touyz, R.M. Bradykinin regulates calpain and proinflammatory signaling through TRPM7-sensitive pathways in vascular smooth muscle cells. Am. J. Physiol. Integr. Comp. Physiol. 2009, 296, R201–R207. [Google Scholar] [CrossRef] [Green Version]
  68. Davis, F.; Azimi, I.; Faville, R.A.; Peters, A.A.; Jalink, K.; Putney, J.; Goodhill, G.; Thompson, E.W.; Roberts-Thomson, S.; Monteith, G. Induction of epithelial–mesenchymal transition (EMT) in breast cancer cells is calcium signal dependent. Oncogene 2013, 33, 2307–2316. [Google Scholar] [CrossRef] [Green Version]
  69. Abiria, S.A.; Krapivinsky, G.; Sah, R.; Santa-Cruz, A.G.; Chaudhuri, D.; Zhang, J.; Adstamongkonkul, P.; DeCaen, P.G.; Clapham, D.E. TRPM7 senses oxidative stress to release Zn2+ from unique intracellular vesicles. Proc. Natl. Acad. Sci. USA 2017, 114, E6079–E6088. [Google Scholar] [CrossRef] [Green Version]
  70. Chokshi, R.; Matsushita, M.; Kozak, J.A. Detailed examination of Mg2+ and pH sensitivity of human TRPM7 channels. Am. J. Physiol. Physiol. 2012, 302, C1004–C1011. [Google Scholar] [CrossRef] [Green Version]
  71. Jahn, R.; Scheller, R.H. SNAREs—Engines for membrane fusion. Nat. Rev. Mol. Cell Biol. 2006, 7, 631–643. [Google Scholar] [CrossRef]
  72. Marshall-Gradisnik, S.; Huth, T.; Chacko, A.; Smith, P.; Staines, D.; Johnston, S. Natural killer cells and single nucleotide polymorphisms of specific ion channels and receptor genes in myalgic encephalomyelitis/chronic fatigue syndrome. Appl. Clin. Genet. 2016, 9, 39–47. [Google Scholar] [CrossRef] [Green Version]
  73. Gavériaux-Ruff, C.; Filliol, D.; Simonin, F.; Matthes, H.W.D.; Kieffer, B.L. Immunosuppression by δ-Opioid Antagonist Naltrindole: δ- and Triple μ/δ/κ-Opioid Receptor Knockout Mice Reveal a Nonopioid Activity. J. Pharmacol. Exp. Ther. 2001, 298, 1193–1198. [Google Scholar]
  74. House, R.V.; Thomas, P.T.; Kozak, J.T.; Bhargava, H.N. Suppression of immune function by non-peptidic delta opioid receptor antagonists. Neurosci. Lett. 1995, 198, 119–122. [Google Scholar] [CrossRef]
  75. Mundra, J.J.; Terskiy, A.; Howells, R.D. Naltrindole inhibits human multiple myeloma cell proliferation in vitro and in a murine xenograft model in vivo. J. Pharmacol. Exp. Ther. 2012, 342, 273–287. [Google Scholar] [CrossRef] [Green Version]
Ijerph 18 11879 g001 550
Figure 1. Baseline NK cell cytotoxicity; n = 17 for HC and n = 17 for ME/CFS. A significantly lower NKCCA was observed in ME/CFS patients compared with HCs at baseline for each effector to target ratio. Data are represented as the mean ± SEM as determined by independent Mann-Whitney U tests. * p < 0.05, *** p < 0.001. Abbreviations: healthy control (HC), myelogenous leukemia target cell line (K562), myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), natural killer (NK), natural killer cell cytotoxic activity (NKCAA).
Figure 1. Baseline NK cell cytotoxicity; n = 17 for HC and n = 17 for ME/CFS. A significantly lower NKCCA was observed in ME/CFS patients compared with HCs at baseline for each effector to target ratio. Data are represented as the mean ± SEM as determined by independent Mann-Whitney U tests. * p < 0.05, *** p < 0.001. Abbreviations: healthy control (HC), myelogenous leukemia target cell line (K562), myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), natural killer (NK), natural killer cell cytotoxic activity (NKCAA).
Ijerph 18 11879 g001
Ijerph 18 11879 g002 550
Figure 2. NK cell cytotoxicity at baseline compared with post-IL-2 stimulation; n = 17 for HC and n = 17 for ME/CFS. (A) 25:1 ratio (B) 12.5:1 ratio (C) 12.5:1 ratio. A significantly higher NKCCA was observed in both HCs and ME/CFS patients following IL-2 (20 IU/mL) stimulation compared with that at baseline for each effector to target ratio. Data are represented as the mean ± SEM as determined by independent Mann-Whitney U tests. * p < 0.05, *** p < 0.001, **** p < 0.0001. Abbreviations: healthy controls (HCs), interleukin-2 (IL-2), myelogenous leukemia target cell line (K562), myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), natural killer (NK), natural killer cell cytotoxic activity (NKCAA).
Figure 2. NK cell cytotoxicity at baseline compared with post-IL-2 stimulation; n = 17 for HC and n = 17 for ME/CFS. (A) 25:1 ratio (B) 12.5:1 ratio (C) 12.5:1 ratio. A significantly higher NKCCA was observed in both HCs and ME/CFS patients following IL-2 (20 IU/mL) stimulation compared with that at baseline for each effector to target ratio. Data are represented as the mean ± SEM as determined by independent Mann-Whitney U tests. * p < 0.05, *** p < 0.001, **** p < 0.0001. Abbreviations: healthy controls (HCs), interleukin-2 (IL-2), myelogenous leukemia target cell line (K562), myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), natural killer (NK), natural killer cell cytotoxic activity (NKCAA).
Ijerph 18 11879 g002
Ijerph 18 11879 g003 550
Figure 3. NK cell cytotoxicity post-IL-2 stimulation compared with TRPM7 drug treatments in HC vs. ME/CFS; n = 17 for HC and n = 17 for ME/CFS. After IL-2 (20 IU/mL) stimulation and TRPM7 agonist (20 IU/mL IL-2 + 25 µM NTB) or antagonist (20 IU/mL IL-2 + 25 µM NTB + 10 µM NS8593) treatments for 24 h, no significant differences were observed within or between groups for each condition for any effector to target ratio. Data are represented as the mean ± SEM as determined by independent Mann-Whitney U tests. Abbreviations: healthy controls (HCs), interleukin-2 (IL-2), myelogenous leukemia target cell line (K562), myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), natural killer (NK), NS8593 (NS), naltriben (NTB).
Figure 3. NK cell cytotoxicity post-IL-2 stimulation compared with TRPM7 drug treatments in HC vs. ME/CFS; n = 17 for HC and n = 17 for ME/CFS. After IL-2 (20 IU/mL) stimulation and TRPM7 agonist (20 IU/mL IL-2 + 25 µM NTB) or antagonist (20 IU/mL IL-2 + 25 µM NTB + 10 µM NS8593) treatments for 24 h, no significant differences were observed within or between groups for each condition for any effector to target ratio. Data are represented as the mean ± SEM as determined by independent Mann-Whitney U tests. Abbreviations: healthy controls (HCs), interleukin-2 (IL-2), myelogenous leukemia target cell line (K562), myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), natural killer (NK), NS8593 (NS), naltriben (NTB).
Ijerph 18 11879 g003
Ijerph 18 11879 g004 550
Figure 4. Co-localization of TRPM7 with actin in NK cells following IL-2 stimulation and treatment with NTB and NS8593 in HC vs. ME/CFS; n = 17 for HC and n = 17 for ME/CFS. The number of cells analyzed per condition are depicted on the bar graphs: n = 165 for HC Control, n = 151 for ME/CFS control, n = 160 for HC NTB, n = 152 for ME/CFS NTB, n = 164 for HC NTB + NS, n = 140 for ME/CFS NTB + NS. A significantly higher co-localization of TRPM7 with actin was identified in the ME/CFS cohort following NS8593 treatment compared with the ME/CFS NTB and HC NS8593 conditions. This difference was not present in the HC cohort. Data are represented as the mean ± SEM as determined by independent Mann-Whitney U tests. ** p < 0.01, *** p < 0.001. Abbreviations: healthy control (HC), myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), natural killer (NK), NS8593 (NS), naltriben (NTB), transient receptor potential melastatin 7 (TRPM7).
Figure 4. Co-localization of TRPM7 with actin in NK cells following IL-2 stimulation and treatment with NTB and NS8593 in HC vs. ME/CFS; n = 17 for HC and n = 17 for ME/CFS. The number of cells analyzed per condition are depicted on the bar graphs: n = 165 for HC Control, n = 151 for ME/CFS control, n = 160 for HC NTB, n = 152 for ME/CFS NTB, n = 164 for HC NTB + NS, n = 140 for ME/CFS NTB + NS. A significantly higher co-localization of TRPM7 with actin was identified in the ME/CFS cohort following NS8593 treatment compared with the ME/CFS NTB and HC NS8593 conditions. This difference was not present in the HC cohort. Data are represented as the mean ± SEM as determined by independent Mann-Whitney U tests. ** p < 0.01, *** p < 0.001. Abbreviations: healthy control (HC), myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), natural killer (NK), NS8593 (NS), naltriben (NTB), transient receptor potential melastatin 7 (TRPM7).
Ijerph 18 11879 g004
Ijerph 18 11879 g005 550
Figure 5. Co-localization of TRPM7 with PIP2 in NK cells following IL-2 stimulation and treatment with NTB and NS8593 in HC vs. ME/CFS; n = 17 for HC and n = 17 for ME/CFS. The number of cells analyzed per condition are depicted on the bar graphs: n = 165 for HC Control, n = 151 for ME/CFS Control, n = 160 for HC NTB, n = 152 for ME/CFS NTB, n = 164 for HC NTB + NS, n = 140 for ME/CFS NTB + NS. A significantly higher co-localization of TRPM7 with PIP2 was identified in the HC and ME/CFS cohorts following NS8593 treatment compared with the HC NTB and ME/CFS NTB conditions, respectively. No significant differences were identified between groups for each condition. Data are presented as the mean ± SEM as determined by independent Mann-Whitney U tests. * p < 0.05, ** p < 0.01. Abbreviations: healthy control (HC), myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), natural killer (NK), NS8593 (NS), phosphoinositide 4,5-bisphosphate (PIP2), transient receptor potential melastatin 7 (TRPM7).
Figure 5. Co-localization of TRPM7 with PIP2 in NK cells following IL-2 stimulation and treatment with NTB and NS8593 in HC vs. ME/CFS; n = 17 for HC and n = 17 for ME/CFS. The number of cells analyzed per condition are depicted on the bar graphs: n = 165 for HC Control, n = 151 for ME/CFS Control, n = 160 for HC NTB, n = 152 for ME/CFS NTB, n = 164 for HC NTB + NS, n = 140 for ME/CFS NTB + NS. A significantly higher co-localization of TRPM7 with PIP2 was identified in the HC and ME/CFS cohorts following NS8593 treatment compared with the HC NTB and ME/CFS NTB conditions, respectively. No significant differences were identified between groups for each condition. Data are presented as the mean ± SEM as determined by independent Mann-Whitney U tests. * p < 0.05, ** p < 0.01. Abbreviations: healthy control (HC), myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), natural killer (NK), NS8593 (NS), phosphoinositide 4,5-bisphosphate (PIP2), transient receptor potential melastatin 7 (TRPM7).
Ijerph 18 11879 g005
Table
Table 1. Participant demographic and full blood count results; n = 17 for HC and n = 17 for ME/CFS. Data are represented as mean ± SEM using Mann-Whitney U tests. *** p < 0.001, **** p < 0.0001. Abbreviations: healthy control (HC), myalgic encephalomyelitis (ME/CFS), body mass index (BMI), World Health Organization disability assessment schedule (WHODAS), short-form health survey (SF-36).
Table 1. Participant demographic and full blood count results; n = 17 for HC and n = 17 for ME/CFS. Data are represented as mean ± SEM using Mann-Whitney U tests. *** p < 0.001, **** p < 0.0001. Abbreviations: healthy control (HC), myalgic encephalomyelitis (ME/CFS), body mass index (BMI), World Health Organization disability assessment schedule (WHODAS), short-form health survey (SF-36).
CategoryItemHCME/CFSp-Value
General demographicsAge (years)48.1 ± 2.148.5 ± 2.00.7264
Gender
Male (%, n)23.5, 523.5, 5
Female (%, n)76.5, 1376.5, 13
BMI (kg/m2)23.9 ± 0.925.2 ± 1.00.3435
WHODASUnderstanding and communication7.1 ± 2.041.6 ± 3.1****
Mobility2.4 ± 1.438.2 ± 6.2****
Self-care0.4 ± 0.418.4 ± 4.9***
Interpersonal relationships3.3 ± 1.733.5 ± 5.0****
Life activities7.4 ± 3.562.5 ± 6.2****
Participation in work/school7.4 ± 3.712.6 ± 5.10.4555
Participation in society4.4 ± 1.952.2 ± 5.1****
Illness demographic
(SF-36)		Pain (%)90.7 ± 3.237.5 ± 5.1****
Physical functioning (%)92.7 ± 4.747.6 ± 6.4****
Role physical (%)93.8 ± 2.725.0 ± 5.5****
General health (%)80.6 ± 2.727.2 ± 3.3****
Social functioning (%)89.7 ± 6.227.2 ± 4.7****
Role emotional (%)95.1 ± 2.463.7 ± 6.9***
Wellbeing74.8 ± 3.543.9 ± 2.8****
Full blood countWhite blood cells6.2 ± 0.55.7 ± 0.30.5454
Lymphocytes1.70 ± 0.151.85 ± 0.090.1243
Neutrophils3.77 ± 0.393.26 ± 0.270.3524
Monocytes0.49 ± 0.050.42 ± 0.020.2778
Eosinophils0.13 ± 0.020.14 ± 0.020.8048
Basophils0.04 ± 0.000.04 ± 0.000.3378
Platelets265 ± 19242 ± 90.2085
Red blood cells4.51 ± 0.094.57 ± 0.120.6645
Hematocrit0.40 ± 0.010.41 ± 0.010.3416
Hemoglobin131 ± 3137 ± 30.2628
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Du Preez, S.; Eaton-Fitch, N.; Cabanas, H.; Staines, D.; Marshall-Gradisnik, S. Characterization of IL-2 Stimulation and TRPM7 Pharmacomodulation in NK Cell Cytotoxicity and Channel Co-Localization with PIP2 in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Patients. Int. J. Environ. Res. Public Health 2021, 18, 11879. https://doi.org/10.3390/ijerph182211879

AMA Style

Du Preez S, Eaton-Fitch N, Cabanas H, Staines D, Marshall-Gradisnik S. Characterization of IL-2 Stimulation and TRPM7 Pharmacomodulation in NK Cell Cytotoxicity and Channel Co-Localization with PIP2 in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Patients. International Journal of Environmental Research and Public Health. 2021; 18(22):11879. https://doi.org/10.3390/ijerph182211879

Chicago/Turabian Style

Du Preez, Stanley, Natalie Eaton-Fitch, Helene Cabanas, Donald Staines, and Sonya Marshall-Gradisnik. 2021. "Characterization of IL-2 Stimulation and TRPM7 Pharmacomodulation in NK Cell Cytotoxicity and Channel Co-Localization with PIP2 in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Patients" International Journal of Environmental Research and Public Health 18, no. 22: 11879. https://doi.org/10.3390/ijerph182211879

APA Style

Du Preez, S., Eaton-Fitch, N., Cabanas, H., Staines, D., & Marshall-Gradisnik, S. (2021). Characterization of IL-2 Stimulation and TRPM7 Pharmacomodulation in NK Cell Cytotoxicity and Channel Co-Localization with PIP2 in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Patients. International Journal of Environmental Research and Public Health, 18(22), 11879. https://doi.org/10.3390/ijerph182211879

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop