Tag: NK cells

Natural killer cytotoxicity in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS): a multi-site clinical assessment of ME/CFS (MCAM) sub-study

Abstract:

Background: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a multisystem illness characterized by substantial reduction in function accompanied by profound unexplained fatigue not significantly relieved by rest, post-exertional malaise, and other symptoms. Reduced natural killer (NK) cell count and cytotoxicity has been investigated as a biomarker for ME/CFS, but few clinical laboratories offer the test and multi-site verification studies have not been conducted.

Methods: We determined NK cell counts and cytotoxicity in 174 (65%) ME/CFS, 86 (32%) healthy control (HC) and 10 (3.7%) participants with other fatigue associated conditions (ill control [IC]) from the Multi-Site Clinical Assessment of ME/CFS (MCAM) study using an assay validated for samples shipped overnight instead of testing on day of venipuncture.

Results: We found a large variation in percent cytotoxicity [mean and (IQR) for ME/CFS and HC respectively, 34.1% (IQR 22.4-44.3%) and 33.6% (IQR 22.9-43.7%)] and no statistically significant differences between patients with ME/CFS and HC (p-value = 0.79). Analysis stratified on illness domain measured with standardized questionnaires did not identify an association of NK cytotoxicity with domain scores. Among all participants, NK cytotoxicity was not associated with survey results of physical and mental well-being, or health factors such as history of infection, obesity, smoking, and co-morbid conditions.

Conclusion: These results indicate this assay is not ready for clinical implementation and studies are needed to further explore immune parameters that may be involved in the pathophysiology of ME/CFS.

Source: Querec TD, Lin JS, Chen Y, Helton B, Kogelnik AM, Klimas NG, Peterson DL, Bateman L, Lapp C, Podell RN, Natelson BH, Unger ER; MCAM Study Group. Natural killer cytotoxicity in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS): a multi-site clinical assessment of ME/CFS (MCAM) sub-study. J Transl Med. 2023 Apr 3;21(1):242. doi: 10.1186/s12967-023-03958-2. PMID: 37013608. https://translational-medicine.biomedcentral.com/articles/10.1186/s12967-023-03958-2 (Full text)

Altered Fatty Acid Oxidation in Lymphocyte Populations of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome

Abstract:

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a disabling multisystem illness in which individuals are plagued with fatigue, inflammatory symptoms, cognitive dysfunction, and the hallmark symptom, post-exertional malaise. While the cause of this disease remains unknown, there is evidence of a potential infectious component that, along with patient symptoms and common onsets of the disease, implicates immune system dysfunction. To further our understanding of the state of ME/CFS lymphocytes, we characterized the role of fatty acids in isolated Natural Killer cells, CD4+ T cells, and CD8+ T cells in circulation and after overnight stimulation, through implicit perturbations to fatty acid oxidation.

We examined samples obtained from at least 8 and as many as 20 subjects for immune cell fatty acid characterization in a variety of experiments and found that all three isolated cell types increased their utilization of lipids and levels of pertinent proteins involved in this metabolic pathway in ME/CFS samples, particularly during higher energy demands and activation. In T cells, we characterized the cell populations contributing to these metabolic shifts, which included CD4+ memory cells, CD4+ effector cells, CD8+ naïve cells, and CD8+ memory cells.

We also discovered that patients with ME/CFS and healthy control samples had significant correlations between measurements of CD4+ T cell fatty acid metabolism and demographic data. These findings provide support for metabolic dysfunction in ME/CFS immune cells. We further hypothesize about the consequences that these altered fuel dependencies may have on T and NK cell effector function, which may shed light on the illness’s mechanism of action.

Source: Maya J, Leddy SM, Gottschalk CG, Peterson DL, Hanson MR. Altered Fatty Acid Oxidation in Lymphocyte Populations of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome. Int J Mol Sci. 2023 Jan 19;24(3):2010. doi: 10.3390/ijms24032010. PMID: 36768336; PMCID: PMC9916395. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9916395/ (Full text)

Impaired TRPM3-dependent calcium influx and restoration using Naltrexone in natural killer cells of myalgic encephalomyelitis/chronic fatigue syndrome patients

Abstract:

Background: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a serious disorder of unknown aetiology. While the pathomechanism of ME/CFS remains elusive, reduced natural killer (NK) cell cytotoxic function is a consistent immunological feature. NK cell effector functions rely on long-term sustained calcium (Ca2+) influx. In recent years evidence of transient receptor potential melastatin 3 (TRPM3) dysfunction supports the hypothesis that ME/CFS is potentially an ion channel disorder. Specifically, reports of single nucleotide polymorphisms, low surface expression and impaired function of TRPM3 have been reported in NK cells of ME/CFS patients. It has been reported that mu (µ)-opioid receptor (µOR) agonists, known collectively as opioids, inhibit TRPM3. Naltrexone hydrochloride (NTX), a µOR antagonist, negates the inhibitory action of µOR on TRPM3 function. Importantly, it has recently been reported that NTX restores impaired TRPM3 function in NK cells of ME/CFS patients.

Methods: Live cell immunofluorescent imaging was used to measure TRPM3-dependent Ca2+ influx in NK cells isolated from n = 10 ME/CFS patients and n = 10 age- and sex-matched healthy controls (HC) following modulation with TRPM3-agonist, pregnenolone sulfate (PregS) and TRPM3-antaognist, ononetin. The effect of overnight (24 h) NTX in vitro treatment on TRPM3-dependent Ca2+ influx was determined.

Results: The amplitude (p < 0.0001) and half-time of Ca2+ response (p < 0.0001) was significantly reduced at baseline in NK cells of ME/CFS patients compared with HC. Overnight treatment of NK cells with NTX significantly improved TRPM3-dependent Ca2+ influx in ME/CFS patients. Specifically, there was no significance between HC and ME/CFS patients for half-time response, and the amplitude of Ca2+ influx was significantly increased in ME/CFS patients (p < 0.0001).

Conclusion: TRPM3-dependent Ca2+ influx was restored in ME/CFS patients following overnight treatment of isolated NK cells with NTX in vitro. Collectively, these findings validate that TRPM3 loss of function results in altered Ca2+ influx supporting the growing evidence that ME/CFS is a TRP ion channel disorder and that NTX provides a potential therapeutic intervention for ME/CFS.

Source: Eaton-Fitch N, Du Preez S, Cabanas H, Muraki K, Staines D, Marshall-Gradisnik S. Impaired TRPM3-dependent calcium influx and restoration using Naltrexone in natural killer cells of myalgic encephalomyelitis/chronic fatigue syndrome patients. J Transl Med. 2022 Feb 16;20(1):94. doi: 10.1186/s12967-022-03297-8. PMID: 35172836. https://translational-medicine.biomedcentral.com/articles/10.1186/s12967-022-03297-8  (Full text)

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

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.

Source: 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 Nov 12;18(22):11879. doi: 10.3390/ijerph182211879. PMID: 34831634. https://pubmed.ncbi.nlm.nih.gov/34831634/

Potential Therapeutic Benefit of Low Dose Naltrexone in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: Role of Transient Receptor Potential Melastatin 3 Ion Channels in Pathophysiology and Treatment

Abstract:

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a debilitating multi-systemic chronic condition of unknown aetiology classified as an immune dysfunction syndrome and neurological disorder. The discovery of the widely expressed Transient Receptor Potential Melastatin 3 (TRPM3) as a nociceptor channel substantially targeted by certain opioid receptors, and its implication in calcium (Ca2+)-dependent Natural Killer (NK) cell immune functions has raised the possibility that TRPM3 may be pharmacologically targeted to treat characteristic symptoms of ME/CFS. Naltrexone hydrochloride (NTX) acts as an antagonist to the mu (μ)-opioid receptor thus negating its inhibitory function on TRPM3.

Based on the benefits reported by patients on their symptoms, low dose NTX (LDN, 3.0–5.0 mg/day) treatment seems to offer some potential benefit suggesting that its effect may be targeted towards the pathomechanism of ME/CFS. As there is no literature confirming the efficacy of LDN for ME/CFS patients in vitro, this study investigates the potential therapeutic effect of LDN in ME/CFS patients. TRPM3 ion channel activity was measured after modulation with Pregnenolone sulfate (PregS) and ononetin in NK cells on 9 ME/CFS patients taking LDN and 9 age- and sex-matched healthy controls using whole-cell patch-clamp technique.

We report that ME/CFS patients taking LDN have restored TRPM3-like ionic currents in NK cells. Small ionic currents with a typical TRPM3-like outward rectification were measured after application of PregS, a TRPM3-agonist, in NK cells from patients taking LDN. Additionally, PregS-evoked ionic currents through TRPM3 were significantly modulated by ononetin, a TRPM3-antagonist, in NK cells from ME/CFS patients taking LDN.

These data support the hypothesis that LDN may have potential as a treatment for ME/CFS by characterising the underlying regulatory mechanisms of LDN treatment involving TRPM3 and opioid receptors in NK cells. Finally, this study may serve for the repurpose of marketed drugs, as well as support the approval of prospective randomized clinical studies on the role and dose of NTX in treating ME/CFS patients.

Source: Helene Cabanas, Katsuhiko Muraki, Natalie Eaton-Fitch, Donald Ross Staines and Sonya Marshall-Gradisnik. Potential Therapeutic Benefit of Low Dose Naltrexone in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: Role of Transient Receptor Potential Melastatin 3 Ion Channels in Pathophysiology and Treatment. Front. Immunol. | https://doi.org/10.3389/fimmu.2021.687806 https://www.frontiersin.org/articles/10.3389/fimmu.2021.687806/full (Full text)

Evaluation of natural killer cell assay performance on shipped blood specimens

Abstract:

Documenting the importance of NK cell function as a biomarker for diseases and physiologic conditions including myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), will require assays amenable to clinical implementation and standardization. Research studies typically perform NK functional assays on the day of sample collection. This pilot study was conducted to compare assay formats and specimen processing to identify those that are most tolerant of conditions required for shipping and amenable to standardization as shown by inter-assay and inter-laboratory correlation of results.

We compared performance within and between assays that measure NK cell function using direct cytotoxicity [chromium-51 release (CRCA) or fluorescence (Flow Cytometry Cytotoxicity Assay, FCCA)] or an indirect surrogate marker (CD107a surface expression)]. Additional variables for within/between assay comparisons included time of testing (same day as specimen collection or next day within 24 h), specimen types [whole blood or isolated peripheral blood mononuclear cells (PBMCs)], and processing method (fresh or cryopreserved). Statistical measures included number of samples tested in assay conditions (n), medians (x͂), interquartile range (IQR), Pearson correlation coefficient (R2), and correlation p-value (p).

Samples came from 3 clinics and included 31 participants. Same day testing was only available for the subset of participants enrolled from the site of the laboratory performing CRCA. Results from same day CRCA testing of whole blood were considered the gold standard [n = 10, x͂=10.0%, IQR = 7.2%], and correlated well with PBMCs isolated next day [n = 26, x͂= 15.6%, IQR = 13.1%] [R2 = 0.59, p = 0.03]. Next day CRCA results were compromised using whole blood or frozen PBMCs. Next day FCCA cytotoxicity in PBMC [n = 30, x͂=34.1%, IQR = 15.5%] correlated with same day CRCA PMBC [R2 = 0.8, p = 0.001] and next day CRCA PMBC [R2 = 0.5, p < 0.0001]. CD107a expression after induction by PMA and ionomycin did not correlate with other cytotoxicity measures. NK function can be measured in PBMCs isolated after overnight shipping/storage at ambient temperature and CRCA and FCCA results on this sample type are well correlated.

Source: Querec TD, Abrams J, Stewart JJ, Barnes Z, Balbin E, Klimas N, Fletcher MA, Brown L, Bertolli J, Unger ER. Evaluation of natural killer cell assay performance on shipped blood specimens. J Immunol Methods. 2021 Apr 2:113049. doi: 10.1016/j.jim.2021.113049. Epub ahead of print. PMID: 33819446. https://pubmed.ncbi.nlm.nih.gov/33819446/

Validation of impaired Transient Receptor Potential Melastatin 3 ion channel activity in natural killer cells from Chronic Fatigue Syndrome/ Myalgic Encephalomyelitis patients

Abstract:

BACKGROUND: Chronic 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.

METHODS: Whole-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.

RESULTS: We 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.

CONCLUSIONS: Impaired 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.

Source: 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 Apr 23;25(1):14. doi: 10.1186/s10020-019-0083-4. https://www.ncbi.nlm.nih.gov/pubmed/31014226

Cellular immune function in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS)

Abstract:

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating condition with unknown aetiology, unclear pathophysiology and with no diagnostic test or biomarker available. Many patients report their ME/CFS began after an acute infection, and subsequent increased frequency of infections, such as colds or influenza, is common. These factors imply an altered immunological status exists in ME/CFS, in at least a proportion of patients, yet previous studies of peripheral immunity have been discrepant and inconclusive.

The UK ME/CFS Biobank, which has collected blood samples from nearly 300 clinically-confirmed ME/CFS patients, enables large-scale studies of immunological function in phenotypically well-characterised participants. In this study, herpes virus serological status and T cell, B cell, NK cell and monocyte populations were investigated in 251 ME/CFS patients, including 54 who were severely affected, and compared with those from 107 healthy participants and with 46 patients with Multiple Sclerosis.

There were no differences in seroprevalence for six human herpes viruses between ME/CFS and healthy controls, although seroprevalence for the Epstein-Barr virus was higher in multiple sclerosis patients. Contrary to previous reports, no significant differences were observed in NK cell numbers, subtype proportions or in vitro responsiveness between ME/CFS patients and healthy control participants. In contrast, the T cell compartment was altered in ME/CFS, with reduced proportions of effector memory CD8+ T cells and of intermediately differentiated CD8+ T cells in ME/CFS. Conversely, there was a significantly increased proportion of mucosal associated invariant T cells (MAIT) cells, especially in severely affected ME/CFS patients.

These abnormalities demonstrate that an altered immunological state does exist in ME/CFS, particularly in severely affected people. This may simply reflect ongoing or recent infection, or may indicate future increased susceptibility to infection. Longitudinal studies of ME/CFS patients are needed to help to determine cause and effect and thus any potential benefits of immuno-modulatory treatments for ME/CFS.

Source: Jacqueline M. Cliff, Elizabeth C. King, Ji-Sook Lee, Nuno Sepulveda, Asia-Sofia Wolf, Caroline Kingdon, Erinna Bowman, Hazel M. Dockrell, Luis C. Nacul, Eliana Lacerda and Eleanor Riley. Cellular immune function in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). Front. Immunol. | doi: 10.3389/fimmu.2019.00796 https://www.frontiersin.org/articles/10.3389/fimmu.2019.00796/full (Full article)

Loss of Transient Receptor Potential Melastatin 3 ion channel function in natural killer cells from Chronic Fatigue Syndrome/Myalgic Encephalomyelitis patients

Abstract:

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.

Source: 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 Aug 14;24(1):44. doi: 10.1186/s10020-018-0046-1.

Longitudinal associations of lymphocyte subsets with clinical outcomes in chronic fatigue syndrome

Abstract:

BACKGROUND: Chronic fatigue syndrome (CFS) is characterized by prolonged fatigue and other physical and neurocognitive symptoms. Some studies suggest that CFS is accompanied by disruptions in the number and function of various lymphocytes. However, it is not clear which lymphocytes might influence CFS symptoms.

PURPOSE: To determine if patient reported fatigue symptoms and physical functioning scores significantly changed across time with lymphocyte counts as evidence of a relation among chronic fatigue symptoms and the immune response.

METHODS: The current longitudinal, naturalistic study assessed the cellular expression of three lymphocyte subtypes — natural killer (NK) cells (CD3-CD16+ and CD3-CD56+) and naïve T cells (CD4+CD45RA+) — to determine whether changes in lymphocytes at 4 time points across 18 months were associated with clinical outcomes, including CFS symptoms, physical functioning, and vitality, among patients with chronic fatigue.. Latent growth curve models were used to examine the longitudinal relationship between lymphocytes and clinical outcomes.

RESULTS: Ninety-three patients with Fukuda-based CFS and seven with non-CFS fatigue provided study data. Results indicated that higher proportions of naïve T cells and lower proportions of NK cells were associated with worse physical functioning, whereas higher proportions of NK cells (CD3-CD16+) and lower proportions of naïve T cells were associated with fewer CFS symptoms.

CONCLUSION: These findings suggest that lymphocytes are modestly related to clinical outcomes over time.

Source: Mehalick ML, Schmaling KB, Sabath DE, Buchwald DS. Longitudinal associations of lymphocyte subsets with clinical outcomes in chronic fatigue syndrome. Fatigue. 2018;6(2):80-91. doi: 10.1080/21641846.2018.1426371. Epub 2018 Jan 12.  https://www.ncbi.nlm.nih.gov/pubmed/30112249