Tag: NK cells

Systems-level temporal immune-metabolic profile in Crimean-Congo hemorrhagic fever virus infection

Abstract:

Crimean-Congo hemorrhagic fever (CCHF) caused by CCHF virus (CCHFV) is one of the epidemic-prone diseases prioritized by the World Health Organisation as public health emergency with an urgent need for accelerated research. The trajectory of host response against CCHFV is multifarious and remains unknown. Here, we reported the temporal spectrum of pathogenesis following the CCHFV infection using genome-wide blood transcriptomics analysis followed by advanced systems biology analysis, temporal immune-pathogenic alterations, and context-specific progressive and postinfection genome-scale metabolic models (GSMM) on samples collected during the acute (T0), early convalescent (T1), and convalescent-phase (T2).

The interplay between the retinoic acid-inducible gene-I-like/nucleotide-binding oligomerization domain-like receptor and tumor necrosis factor signaling governed the trajectory of antiviral immune responses. The rearrangement of intracellular metabolic fluxes toward the amino acid metabolism and metabolic shift toward oxidative phosphorylation and fatty acid oxidation during acute CCHFV infection determine the pathogenicity. The upregulation of the tricarboxylic acid cycle during CCHFV infection, compared to the noninfected healthy control and between the severity groups, indicated an increased energy demand and cellular stress. The upregulation of glycolysis and pyruvate metabolism potentiated energy generation through alternative pathways associated with the severity of the infection.

The downregulation of metabolic processes at the convalescent phase identified by blood cell transcriptomics and single-cell type proteomics of five immune cells (CD4+ and CD8+ T cells, CD14+ monocytes, B cells, and NK cells) potentially leads to metabolic rewiring through the recovery due to hyperactivity during the acute phase leading to post-viral fatigue syndrome.

Source: Ambikan AT, Elaldi N, Svensson-Akusjärvi S, Bagci B, Pektas AN, Hewson R, Bagci G, Arasli M, Appelberg S, Mardinoglu A, Sood V, Végvári Á, Benfeitas R, Gupta S, Cetin I, Mirazimi A, Neogi U. Systems-level temporal immune-metabolic profile in Crimean-Congo hemorrhagic fever virus infection. Proc Natl Acad Sci U S A. 2023 Sep 12;120(37):e2304722120. doi: 10.1073/pnas.2304722120. Epub 2023 Sep 5. PMID: 37669378; PMCID: PMC10500270. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10500270/ (Full text)

Investigation into the restoration of TRPM3 ion channel activity in post-COVID-19 condition: a potential pharmacotherapeutic target

Abstract:

Introduction: Recently, we reported that post COVID-19 condition patients also have Transient Receptor Potential Melastatin 3 (TRPM3) ion channel dysfunction, a potential biomarker reported in natural killer (NK) cells from Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) patients. As there is no universal treatment for post COVID-19 condition, knowledge of ME/CFS may provide advances to investigate therapeutic targets. Naltrexone hydrochloride (NTX) has been demonstrated to be beneficial as a pharmacological intervention for ME/CFS patients and experimental investigations have shown NTX restored TRPM3 function in NK cells. This research aimed to: i) validate impaired TRPM3 ion channel function in post COVID-19 condition patients compared with ME/CFS; and ii) investigate NTX effects on TRPM3 ion channel activity in post COVID-19 condition patients.

Methods: Whole-cell patch-clamp was performed to characterize TRPM3 ion channel activity in freshly isolated NK cells of post COVID-19 condition (N = 9; 40.56 ± 11.26 years), ME/CFS (N = 9; 39.33 ± 9.80 years) and healthy controls (HC) (N = 9; 45.22 ± 9.67 years). NTX effects were assessed on post COVID-19 condition (N = 9; 40.56 ± 11.26 years) and HC (N = 7; 45.43 ± 10.50 years) where NK cells were incubated for 24 hours in two protocols: treated with 200 µM NTX, or non-treated; TRPM3 channel function was assessed with patch-clamp protocol.

Results: This investigation confirmed impaired TRPM3 ion channel function in NK cells from post COVID-19 condition and ME/CFS patients. Importantly, PregS-induced TRPM3 currents were significantly restored in NTX-treated NK cells from post COVID-19 condition compared with HC. Furthermore, the sensitivity of NK cells to ononetin was not significantly different between post COVID-19 condition and HC after treatment with NTX.

Discussion: Our findings provide further evidence identifying similarities of TRPM3 ion channel dysfunction between ME/CFS and post COVID-19 condition patients. This study also reports, for the first time, TRPM3 ion channel activity was restored in NK cells isolated from post COVID-19 condition patients after in vitro treatment with NTX. The TRPM3 restoration consequently may re-establish TRPM3-dependent calcium (Ca2+) influx. This investigation proposes NTX as a potential therapeutic intervention and TRPM3 as a treatment biomarker for post COVID-19 condition.

Source: Etianne Martini Sasso, Katsuhiko Muraki, Natalie Eaton-Fitch, Peter Smith, Andrew Jeremijenko, Paul Griffin, Sonya Marshall-Gradisnik. Investigation into the restoration of TRPM3 ion channel activity in post-COVID-19 condition: a potential pharmacotherapeutic target. Front. Immunol., 02 May 2024; Sec. Multiple Sclerosis and Neuroimmunology; Volume 15 – 2024 | https://doi.org/10.3389/fimmu.2024.1264702. https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2024.1264702/full (Full text)

SARS-CoV-2 viral persistence in lung alveolar macrophages is controlled by IFN-γ and NK cells

Abstract:

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA generally becomes undetectable in upper airways after a few days or weeks postinfection. Here we used a model of viral infection in macaques to address whether SARS-CoV-2 persists in the body and which mechanisms regulate its persistence.

Replication-competent virus was detected in bronchioalveolar lavage (BAL) macrophages beyond 6 months postinfection. Viral propagation in BAL macrophages occurred from cell to cell and was inhibited by interferon-γ (IFN-γ). IFN-γ production was strongest in BAL NKG2r+CD8+ T cells and NKG2Alo natural killer (NK) cells and was further increased in NKG2Alo NK cells after spike protein stimulation.

However, IFN-γ production was impaired in NK cells from macaques with persisting virus. Moreover, IFN-γ also enhanced the expression of major histocompatibility complex (MHC)-E on BAL macrophages, possibly inhibiting NK cell-mediated killing. Macaques with less persisting virus mounted adaptive NK cells that escaped the MHC-E-dependent inhibition.

Our findings reveal an interplay between NK cells and macrophages that regulated SARS-CoV-2 persistence in macrophages and was mediated by IFN-γ.

Source: Huot N, Planchais C, Rosenbaum P, Contreras V, Jacquelin B, Petitdemange C, Lazzerini M, Beaumont E, Orta-Resendiz A, Rey FA, Reeves RK, Le Grand R, Mouquet H, Müller-Trutwin M. SARS-CoV-2 viral persistence in lung alveolar macrophages is controlled by IFN-γ and NK cells. Nat Immunol. 2023 Nov 2. doi: 10.1038/s41590-023-01661-4. Epub ahead of print. PMID: 37919524. https://www.nature.com/articles/s41590-023-01661-4 (Full text)

Surveying the Metabolic and Dysfunctional Profiles of T Cells and NK Cells in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome

Abstract:

Millions globally suffer from myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). The inflammatory symptoms, illness onset, recorded outbreak events, and physiological variations provide strong indications that ME/CFS, at least sometimes, has an infectious origin, possibly resulting in a chronic unidentified viral infection.
Meanwhile, studies exposing generalized metabolic disruptions in ME/CFS have stimulated interest in isolated immune cells with an altered metabolic state. As the metabolism dictates the cellular function, dissecting the biomechanics of dysfunctional immune cells in ME/CFS can uncover states such as exhaustion, senescence, or anergy, providing insights into the consequences of these phenotypes in this disease. Despite the similarities that are seen metabolically between ME/CFS and other chronic viral infections that result in an exhausted immune cell state, immune cell exhaustion has not yet been verified in ME/CFS.
This review explores the evidence for immunometabolic dysfunction in ME/CFS T cell and natural killer (NK) cell populations, comparing ME/CFS metabolic and functional features to dysfunctional immune cell states, and positing whether anergy, exhaustion, or senescence could be occurring in distinct immune cell populations in ME/CFS, which is consistent with the hypothesis that ME/CFS is a chronic viral disease.
This comprehensive review of the ME/CFS immunometabolic literature identifies CD8+ T cell exhaustion as a probable contender, underscores the need for further investigation into the dysfunctional state of CD4+ T cells and NK cells, and explores the functional implications of molecular findings in these immune-cell types. Comprehending the cause and impact of ME/CFS immune cell dysfunction is critical to understanding the physiological mechanisms of ME/CFS, and developing effective treatments to alleviate the burden of this disabling condition.
Source: Maya J. Surveying the Metabolic and Dysfunctional Profiles of T Cells and NK Cells in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome. International Journal of Molecular Sciences. 2023; 24(15):11937. https://doi.org/10.3390/ijms241511937 https://www.mdpi.com/1422-0067/24/15/11937 (Full text)

Altered TRPM7-Dependent Calcium Influx in Natural Killer Cells of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Patients

Abstract:

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a disabling multisystemic condition. The pathomechanism of ME/CFS remains unestablished; however, impaired natural killer (NK) cell cytotoxicity is a consistent feature of this condition. Calcium (Ca2+) is crucial for NK cell effector functions.
Growing research recognises Ca2+ signalling dysregulation in ME/CFS patients and implicates transient receptor potential ion channel dysfunction. TRPM7 (melastatin) was recently considered in the pathoaetiology of ME/CFS as it participates in several Ca2+-dependent processes that are central to NK cell cytotoxicity which may be compromised in ME/CFS. TRPM7-dependent Ca2+ influx was assessed in NK cells isolated from n = 9 ME/CFS patients and n = 9 age- and sex-matched healthy controls (HCs) using live cell fluorescent imaging techniques.
Slope (p < 0.05) was significantly reduced in ME/CFS patients compared with HCs following TRPM7 activation. Half-time of maximal response (p < 0.05) and amplitude (p < 0.001) were significantly reduced in the HCs compared with the ME/CFS patients following TRPM7 desensitisation.
Findings from this investigation suggest that TRPM7-dependent Ca2+ influx is reduced with agonism and increased with antagonism in ME/CFS patients relative to the age- and sex-matched HCs. The outcomes reported here potentially reflect TRPM3 dysfunction identified in this condition suggesting that ME/CFS is a TRP ion channelopathy.
Source: Du Preez S, Eaton-Fitch N, Smith PK, Marshall-Gradisnik S. Altered TRPM7-Dependent Calcium Influx in Natural Killer Cells of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Patients. Biomolecules. 2023; 13(7):1039. https://doi.org/10.3390/biom13071039 https://www.mdpi.com/2218-273X/13/7/1039 (Full text)

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)