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. 2017 Feb;187(2):284-293.
doi: 10.1111/cei.12882. Epub 2016 Nov 23.

Impaired calcium mobilization in natural killer cells from chronic fatigue syndrome/myalgic encephalomyelitis patients is associated with transient receptor potential melastatin 3 ion channels

T Nguyen  1   2 , S Johnston  1   2 , L Clarke  1   2 , P Smith  1 , D Staines  1   2 , S Marshall-Gradisnik  1   2
Affiliations

Impaired calcium mobilization in natural killer cells from chronic fatigue syndrome/myalgic encephalomyelitis patients is associated with transient receptor potential melastatin 3 ion channels

T Nguyen et al. Clin Exp Immunol. 2017 Feb.

Abstract

Transient receptor potential melastatin subfamily 3 (TRPM3) ion channels play a role in calcium (Ca2+ ) cell signalling. Reduced TRPM3 protein expression has been identified in chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) patients. However, the significance of TRPM3 and association with intracellular Ca2+ mobilization has yet to be determined. Fifteen CFS/ME patients (mean age 48·82 ± 9·83 years) and 25 healthy controls (mean age 39·2 ± 12·12 years) were examined. Isolated natural killer (NK) cells were labelled with fluorescent antibodies to determine TRPM3, CD107a and CD69 receptors on CD56dim CD16+ NK cells and CD56bright CD16dim/- NK cells. Ca2+ flux and NK cytotoxicity activity was measured under various stimulants, including pregnenolone sulphate (PregS), thapsigargin (TG), 2-aminoethoxydiphenyl borate (2APB) and ionomycin. Unstimulated CD56bright CD16dim/- NK cells showed significantly reduced TRPM3 receptors in CFS/ME compared with healthy controls (HC). Ca2+ flux showed no significant difference between groups. Moreover, PregS-stimulated CD56bright CD16dim/- NK cells showed a significant increase in Ca2+ flux in CFS/ME patients compared with HC. By comparison, unstimulated CD56dim CD16+ NK cells showed no significant difference in both Ca2+ flux and TRPM3 expression. PregS-stimulated CD56dim CD16+ NK cells increased TRPM3 expression significantly in CFS/ME, but this was not associated with a significant increase in Ca2+ flux. Furthermore, TG-stimulated CD56dim CD16+ NK cells increased K562 cell lysis prior to PregS stimulation in CFS/ME patients compared with HC. Differential expression of TRPM3 and Ca2+ flux between NK cell subtypes may provide evidence for their role in the pathomechanism involving NK cell cytotoxicity activity in CFS/ME.

Keywords: cell surface molecules; inhibitory/activating receptors; natural killer cells.

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Figures

Figure 1
Figure 1
Systematic representation of calcium ions mobilization. Ionomycin is an ionopore that allows extracellular calcium ion into the cytosol. 2‐APB is a non‐selective TRPM and IP3R receptor, whereas thapsigargin inhibits intracellular calcium stores replenishment. ER = endoplasmic reticulum; SOC = store‐operated calcium channel; CRAC = calcium release activated channel; TRPM3 = transient receptor potential melastatin subfamily 3; STIM = stromal interaction molecule; SERCA = sarco/endoplasmic reticulum Ca2+‐ATPase; IP3R = inositol trisphosphate receptor; PregS = pregnenolone sulphate; 2‐APB = 2‐aminoethoxydiphenyl borate; Ca2+ = calcium.
Figure 2
Figure 2
Natural killer (NK) cell TRPM3 expression in HC and CFS/ME patients. CD56brightCD16dim/– NK cells expressed TRPM3 as cells/ml under various conditions. (a) Unstimulated PregS and ionomycin. (b) TG and TG + PregS. (c) 2APB and 2APB + PregS. CD56dimCD16+ NK cells expressed TRPM3 as cells/ml under various conditions. (d) PregS and ionomycin. (e) TG and TG + PregS. (f) 2APB and 2APB + PregS. Data are represented as mean ± standard error of the mean. Asterisks (*) and (**) represent statistical significance at P < 0·05 and P < 0·01, respectively. US = unstimulated; PregS = pregnenolone sulphate; TG = thapsigargin; HC = healthy controls; 2‐APB = 2‐aminoethoxydiphenyl borate; CFS/ME = chronic fatigue syndrome/myalgic encephalomyelitis. [Colour figure can be viewed at wileyonlinelibrary.com]
Figure 3
Figure 3
FlowJo analysis of Ca2+ mobilization response in isolated natural killer (NK) cells. (a) Gating strategies for CD56brightCD16dim/– NK cells and CD56dimCD16+ NK cells are shown as a pseudocolour dot‐plot. (b) NK cell Ca2+ flux is response over time, and displayed as a pseudocolour dot‐plot. (c) Summary of the kinetic analysis of the Ca2+ flux in NK cells. Peak t is the time that the maximum y‐axis value occurred for the specific time range noted. Peak is the magnitude of the y‐axis value at its maximum for the specific time range noted. The mean of the y‐axis (mean Y) value is for the time range noted. The slope is the gain or loss of intensity over the duration of the time range for the calculated linear regression line of the data in this range. The area under the curve (AUC) is indicated by the grey stripes. Background of the calcium curve is shaded in pink. Post‐stimulant calcium response curve is shaded in purple.
Figure 4
Figure 4
Cytoplasmic calcium in natural killer (NK) cells from HC and CFS/ME patients. (a) CD56bright CD16dim/– NK cell calcium flux response area under the curve. (b) CD56dimCD16+TRPM3+ NK cell calcium flux response area under the curve. Data are represented as mean ± standard error of the mean. Asterisks (*) and (**) represent statistical significance at P < 0·05 and P < 0·01, respectively. Abbreviations: US = unstimulated; PregS = pregnenolone sulphate; TG = thapsigargin; HC = healthy controls; CFS/ME = chronic fatigue syndrome/myalgic encephalomyelitis.
Figure 5
Figure 5
Natural killer (NK) cell cytotoxic activity after incubation with ionomycin, PregS, TG + PregS and 2APB + PregS in HC and CFS/ME. Note significant elevation of K562 cell death in CFS/ME following TG + PregS. Data are represented as mean ± standard error of the mean. Asterisk (*) represents statistical significance at P < 0·05. PregS = pregnenolone sulphate; 2‐APB = 2‐aminoethoxydiphenyl borate; TG = thapsigargin; CFS/ME = chronic fatigue syndrome/myalgic encephalomyelitis.
Figure 6
Figure 6
A conceptual natural killer (NK) model for chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) patients. Pregnenolone sulphate (PregS) is a potent steroid that activated transient receptor potential melastatin 3 ion channels (TRPM3) on NK cell subsets. PregS in combination with thapsigargin elevated cytoplasmic calcium and is suggested to phosphorylate extracellular‐regulated kinase (ERK) 1/2 and polarization of secretory granules for degranulation in CD56dimCD16+ NK cells, resulting in target K562 cell death, whereas PregS‐stimulated CD56brightCD16dim/– NK cells may activate p38 MAPK signalling pathways involved in the nuclear factor kappa B (NF‐κβ) inflammatory immune response.
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