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

Abstract Background Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a serious multifactorial disorder. The origin remains ambiguous, however reduced natural killer (NK) cell cytotoxicity is a consistent immunological feature of ME/CFS. Impaired transient receptor potential melastatin 3 (TRPM3), a phosphatidylinositol dependent channel, and impaired calcium mobilisation have been implicated in ME/CFS pathology. This investigation aimed to examine the localisation of TRPM3 at the NK cell plasma membrane and co-localisation with phosphatidylinositol 4,5-bisphosphate (PIP2). The effect of IL-2 priming and treatment using pregnenolone sulfate (PregS) and ononetin on TRPM3 co-localisation and NK cell cytotoxicity in ME/CFS patients and healthy controls (HC) was also investigated. Methods NK cells were isolated from 15 ME/CFS patients and 15 age- and sex-matched HC. Immunofluorescent technique was used to determine co-localisation of TRPM3 with the NK cell membrane and with PIP2 of ME/CFS patients and HC. Flow cytometry was used to determine NK cell cytotoxicity. Following IL-2 stimulation and treatment with PregS and ononetin changes in co-localisation and NK cell cytotoxicity were measured. Results Overnight treatment of NK cells with PregS and ononetin resulted in reduced co-localisation of TRPM3 with PIP2 and actin in HC. Co-localisation of TRPM3 with PIP2 in NK cells was significantly reduced in ME/CFS patients compared with HC following priming with IL-2. A significant increase in co-localisation of TRPM3 with PIP2 was reported following overnight treatment with ononetin within ME/CFS patients and between groups. Baseline NK cell cytotoxicity was significantly reduced in ME/CFS patients; however, no changes were observed following overnight incubation with IL-2, PregS and ononetin between HC and ME/CFS patients. IL-2 stimulation significantly enhanced NK cell cytotoxicity in HC and ME/CFS patients. Conclusion Significant changes in co-localisation suggest PIP2-dependent TRPM3 function may be impaired in ME/CFS patients. Stimulation of NK cells with IL-2 significantly enhanced cytotoxic function in ME/CFS patients demonstrating normal function compared with HC. A crosstalk exists between IL-2 and TRPM3 intracellular signalling pathways which are dependent on Ca2+ influx and PIP2. While IL-2R responds to IL-2 binding in vitro, Ca2+ dysregulation and impaired intracellular signalling pathways impede NK cell function in ME/CFS patients.

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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 ◽

10.3390/ijerph182211879 ◽

2021 ◽

Vol 18 (22) ◽

pp. 11879

Author(s):

Stanley Du Preez ◽

Natalie Eaton-Fitch ◽

Helene Cabanas ◽

Donald Staines ◽

Sonya Marshall-Gradisnik

Keyword(s):

Chronic Fatigue Syndrome ◽

Nk Cells ◽

Nk Cell ◽

Expression Patterns ◽

Chronic Fatigue ◽

Myalgic Encephalomyelitis ◽

Cell Cytotoxicity ◽

Fatigue Syndrome ◽

Nk Cell Cytotoxicity

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.

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Transient receptor potential melastatin 2 channels are overexpressed in myalgic encephalomyelitis/chronic fatigue syndrome patients

Journal of Translational Medicine ◽

10.1186/s12967-019-02155-4 ◽

2019 ◽

Vol 17 (1) ◽

Author(s):

Cassandra Balinas ◽

Helene Cabanas ◽

Donald Staines ◽

Sonya Marshall-Gradisnik

Keyword(s):

Nk Cells ◽

Transient Receptor Potential ◽

Nk Cell ◽

Receptor Potential ◽

Surface Expression ◽

Chronic Fatigue ◽

Cell Cytotoxicity ◽

Fatigue Syndrome ◽

Transient Receptor Potential Melastatin ◽

Nk Cell Cytotoxicity

Abstract Background Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is hallmarked by a significant reduction in natural killer (NK) cell cytotoxicity, a mechanism tightly regulated by calcium (Ca2+). Interestingly, interleukin-2 (IL-2) increases NK cell cytotoxicity. Transient receptor potential melastatin 2 (TRPM2) ion channels are fundamental for Ca2+ signalling in NK cells. This pilot investigation aimed to characterise TRPM2 and CD38 surface expression in vitro on NK cells in ME/CFS patients. This investigation furthermore examined the pharmaceutical effect of 8-bromoadenosine phosphoribose (8-Br-ADPR) and N6-Benzoyladenosine-3′,5′-cyclic monophosphate (N6-Bnz-cAMP) on TRPM2 and CD38 surface expression and NK cell cytotoxicity between ME/CFS and healthy control (HC) participants. Methods Ten ME/CFS patients (43.45 ± 12.36) and 10 HCs (43 ± 12.27) were age and sex-matched. Isolated NK cells were labelled with fluorescent antibodies to determine baseline and drug-treated TRPM2 and CD38 surface expression on NK cell subsets. Following IL-2 stimulation, NK cell cytotoxicity was measured following 8-Br-ADPR and N6-Bnz-cAMP drug treatments by flow cytometry. Results Baseline TRPM2 and CD38 surface expression was significantly higher on NK cell subsets in ME/CFS patients compared with HCs. Post IL-2 stimulation, TRPM2 and CD38 surface expression solely decreased on the CD56DimCD16+ subset. 8-Br-ADPR treatment significantly reduced TRPM2 surface expression on the CD56BrightCD16Dim/− subset within the ME/CFS group. Baseline cell cytotoxicity was significantly reduced in ME/CFS patients, however no changes were observed post drug treatment in either group. Conclusion Overexpression of TRPM2 on NK cells may function as a compensatory mechanism to alert a dysregulation in Ca2+ homeostasis to enhance NK cell function in ME/CFS, such as NK cell cytotoxicity. As no improvement in NK cell cytotoxicity was observed within the ME/CFS group, an impairment in the TRPM2 ion channel may be present in ME/CFS patients, resulting in alterations in [Ca2+]i mobilisation and influx, which is fundamental in driving NK cell cytotoxicity. Differential expression of TRPM2 between NK cell subtypes may provide evidence for their role in the pathomechanism involving NK cell cytotoxicity activity in ME/CFS.

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A systematic review of natural killer cells profile and cytotoxic function in myalgic encephalomyelitis/chronic fatigue syndrome

Systematic Reviews ◽

10.1186/s13643-019-1202-6 ◽

2019 ◽

Vol 8 (1) ◽

Cited By ~ 4

Author(s):

Natalie Eaton-Fitch ◽

Stanley du Preez ◽

Hélène Cabanas ◽

Donald Staines ◽

Sonya Marshall-Gradisnik

Keyword(s):

Nk Cells ◽

Cytokine Production ◽

Nk Cell ◽

Chronic Fatigue ◽

Cell Phenotype ◽

Cell Cytotoxicity ◽

Protein Levels ◽

Fatigue Syndrome ◽

Nk Cell Cytotoxicity ◽

Cytotoxic Function

Abstract Background Compromised natural killer (NK) cell cytotoxic function is a well-documented and consistent feature of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). Other outcomes evaluated in NK cells of ME/CFS patients, however, remain equivocal. The aim of this study was to conduct a systematic review of the literature regarding NK cell phenotype, receptor expression, cytokine production and cytotoxicity in ME/CFS patients and determine the appropriateness as a model for ME/CFS. Methods Medline (EBSCOHost), Scopus, EMBASE and PubMed databases were systematically searched to source relevant papers published between 1994 and March 2018. This review included studies examining NK cells’ features in ME/CFS patients compared with HC following administration of specific inclusion and exclusion criteria. Secondary outcomes included genetic analysis in isolated NK cells or quality of life assessment. Quality assessment was completed using the Downs and Black checklist in addition to The Joanna Briggs Institute checklist. Results Seventeen eligible publications were included in this review. All studies were observational case control studies. Of these, 11 investigated NK cell cytotoxicity, 14 investigated NK cell phenotype and receptor profiles, three examined NK cell cytokine production, six investigated NK cell lytic protein levels and four investigated NK cell degranulation. Impaired NK cell cytotoxicity remained the most consistent immunological report across all publications. Other outcomes investigated differed between studies. Conclusion A consistent finding among all papers included in this review was impaired NK cell cytotoxicity, suggesting that it is a reliable and appropriate cellular model for continued research in ME/CFS patients. Aberrations in NK cell lytic protein levels were also reported. Although additional research is recommended, current research provides a foundation for subsequent investigations. It is possible that NK cell abnormalities can be used to characterise a subset of ME/CFS due to the heterogeneity of both the illness itself and findings between studies investigating specific features of NK function.

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Pilot assessment of low NK cell-mediated ADCC and FCGR3A genetics in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS): Based on inclusion of family members without ME/CFS as controls, low ADCC is unsuitable as a diagnostic biomarker

10.1101/2019.12.20.19015438 ◽

2019 ◽

Author(s):

Alexander P. Sung ◽

Jennifer J-J Tang ◽

Michael J. Guglielmo ◽

Julie Smith-Gagen ◽

Lucinda Bateman ◽

...

Keyword(s):

Chronic Fatigue Syndrome ◽

Nk Cells ◽

Effector Cell ◽

Nk Cell ◽

Family Members ◽

Chronic Fatigue ◽

Healthy Controls ◽

Diagnostic Biomarker ◽

Fatigue Syndrome ◽

Cell Counts

AbstractADCC (antibody-dependent cell-mediated cytotoxicity) is dependent on the varying capacity of NK cells to kill, the affinities of FCGR3A-encoded CD16A receptors for antibody, and the presence of antigen-specific antibodies. In vivo ADCC depends on the number of CD16A receptor-positive NK cells in blood. We hypothesized that low ADCC cell function or low effector cell numbers could be biomarkers or risk factors for myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). We measured NK cell ADCC lytic capacity and antibody recognition, CD16Apositive NK cells/µl blood, and FCGR3A homozygosity for the F allele that encodes low affinity CD16A antibody receptors. ME/CFS patients met the Fukuda 1994 diagnostic criteria. In this pilot report, we examined 5 families, each with 2 to 5 ME/CFS patients, and compared 11 patients, 22 family members without ME/CFS, and 16 unrelated healthy controls. ADCC was measured as CX1:1 cytotoxic capacity (the percentage of 51Cr-Daudi tumors with obinutuzumab anti-CD20 antibody that were killed at a 1:1 ratio of CD16Apos NKs to Daudis) and CX-slope. Individual CX1:1 capacities varied from 16.2% to 81.8% and were comparable between patients and unaffected family members, while the ADCC of both family groups was lower than the unrelated healthy controls. The lack of difference between patients and their unaffected family members indicates that low ADCC is unsuitable as a diagnostic biomarker for ME/CFS. Familial CD16Apos NK blood cell counts were lower than unrelated healthy controls. The potential for synergistic effects of combined low CX1:1 and low effector cell counts occurring in the same individual was 24-fold greater for CFS family members than for unrelated controls. FCGR3A of the families was predominantly F/F homozygous, correlating with the observed low EC50 for NK recognition of target cell-bound antibody. In summary, low ADCC is unsuitable as a biomarker, but could be a familial risk factor, for ME/CFS.

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Normal Salivary Cortisol and NK Cell Function in Adolescents With Chronic Fatigue Syndrome Following Infectious Mononucleosis

Archives of Pediatric Infectious Diseases ◽

10.5812/pedinfect.13107 ◽

2013 ◽

Vol 2 (4) ◽

pp. 211-216 ◽

Cited By ~ 5

Author(s):

Ben Z Katz ◽

Donald Zimmerman ◽

Maurice RG Gorman ◽

Cynthia J Mears ◽

Yukiko Shiraishi ◽

...

Keyword(s):

Chronic Fatigue Syndrome ◽

Infectious Mononucleosis ◽

Salivary Cortisol ◽

Cell Function ◽

Nk Cell ◽

Chronic Fatigue ◽

Fatigue Syndrome

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Applying CRISPR-based genetic screens to identify drivers of tumor-cell sensitivity towards NK-cell attack

10.1101/531962 ◽

2019 ◽

Cited By ~ 1

Author(s):

Klara Klein ◽

Tim Wang ◽

Eric S. Lander ◽

Marcus Altfeld ◽

Wilfredo F. Garcia-Beltran

Keyword(s):

Tumor Cell ◽

Nk Cells ◽

Tumor Cells ◽

Cell Function ◽

Nk Cell ◽

Target Cells ◽

Cell Cytotoxicity ◽

Genetic Screens ◽

A Genome ◽

Nk Cell Cytotoxicity

ABSTRACTNatural killer (NK) cells distinguish cancer cells from healthy cells using an array of germline-encoded receptors that interact with ligands expressed on target cells. A balance of inhibitory and activating signals transduced by these receptors regulate NK cell function to provide anti-tumor immunity while maintaining self-tolerance. However, knowledge of the spectrum of factors regulating NK-cell-mediated cytotoxicity, including the contribution of specific ligands and regulatory mechanisms for their expression on tumor cells, remains incomplete. Here, we apply a genome-wide loss-of-function screen in tumor cells using CRISPR/Cas9 technology to identify the factors that promote NK-cell cytotoxicity towards tumor cells. We established the drivers of tumor-cell sensitivity towards NK-cell attack (TuSeNKA) screening approach using the chronic myeloid leukemia (CML) cell line, K562. Interestingly, we identified B7H6, the ligand for the activating NK cell receptor NKp30, as the single factor whose loss resulted in increased resistance of K562 cells towards NK cells. Our study shows that combination of CRISPR-based genetic screens with NK-cell cytotoxicity assays is a valuable tool for identifying functionally relevant NK cell-tumor cell interactions, paving the way for further investigations that unravel the complexity of signals that promote NK-cell recognition of transformed cells and develop therapies that target these modes of tumor-cell killing.

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Ibrutinib Potentiated NK Cell-Mediated Cytotoxicity in Mouse Models of B-Cell Lymphomas

Blood ◽

10.1182/blood.v128.22.4140.4140 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4140-4140 ◽

Cited By ~ 1

Author(s):

Hsu-Ping Kuo ◽

Sidney Hsieh ◽

Jennifer Whang ◽

Yujun Huang ◽

Mint Sirisawad ◽

...

Keyword(s):

B Cell ◽

Nk Cells ◽

Mouse Models ◽

Cell Function ◽

Nk Cell ◽

Cell Lymphoma ◽

Cell Cytotoxicity ◽

Nsg Mice ◽

Equity Ownership ◽

Nk Cell Cytotoxicity

Abstract Background: Ibrutinib, a first-in-class, once-daily oral inhibitor of Bruton's tyrosine kinase (BTK), is indicated by the US FDA for the treatment of patients with chronic lymphocytic leukemia/small lymphocytic lymphoma, including patients with deletion 17p, patients with mantle cell lymphoma who have received at least 1 prior therapy, and patients with Waldenström's macroglobulinemia. In addition to blocking B-cell activation via inhibition of BTK signaling, ibrutinib was reported to modulate immune function by driving TH1 response through IL-2-inducible T-cell kinase (Dubovsky, Blood 2013). Ibrutinib was also shown to reprogram macrophages toward a TH1 phenotype that fostered CD8+ T-cell cytotoxicity in pancreas ductal adenocarcinoma-bearing mice (Gunderson, Cancer Discov 2016). Both the direct and indirect effects on TH1 response may play a role in the therapeutic efficacy of ibrutinib in models of lymphoma and solid tumors (Sagiv-Barfi, PNAS 2015). Another essential component of the immune system with a role against cancer is natural killer (NK) cells involved in the recognition and elimination of tumor cells. In this study, we evaluated the effects of ibrutinib on NK cell-mediated cytotoxicity in mouse models of B-cell lymphoma. Methods: Experiments in TMD8 xenograft models were performed in CB17.SCID and NSG mice, and experiments in an A20 syngeneic model were performed in BALB/c mice. Mice were orally administered ibrutinib (once daily) for a total duration of 2 weeks starting from the time tumors reached a volume of 100-150 mm3. Tumor BTK occupancy was determined by a gel-based probe assay 4 hours after the last dose of ibrutinib, and was normalized to the total BTK level. NK cell-mediated cytotoxicity was also evaluated in mice carrying X-linked spontaneous mutation in BTK, Btk(xid) mice (009361; Jackson Laboratory). For the NK cell cytotoxicity assay, mouse splenocytes were added to PKH67-labeled YAC-1 cells at different effector-to-target ratios and incubated at 37°C for 4 hours. Propidium iodide was added and flow cytometry was performed to determine target cell viability. Human or mouse cytokines/chemokines were quantified using MILLIPLEX® MAP Kit (HCYTMAG-60K-PX38 and MCYTMAG-70K-PX32). Results: In TMD8 ABC-DLBCL models, tumor suppression after ibrutinib treatment was only observed in CB17.SCID mice but not in NSG mice despite tumor BTK occupancy of >90% in both models. As these 2 strains of mice differ in their NK cell profile (NSG mice lack mature T, B, and NK cells whereas CB17.SCID mice are severely deficient in T and B cells), we were interested in studying the immune modulation function of ibrutinib on NK cells. Interestingly, elevated NK cell cytotoxicity was seen in Btk(xid) mice compared to Btk wild-type mice, suggesting a potential role of BTK depletion in NK cell function. Treatment with ibrutinib increased NK cell-mediated cytotoxicity in both syngeneic A20 B-cell lymphoma and TMD8 xenograft models (Figure 1), but did not affect NK cell population (% of NKp46+ or CD3-CD49b+). A20 tumor-bearing mice had lower NK cell cytotoxicity compared to non-tumor-bearing mice, suggesting suppression of NK cell function by tumors. In addition, ibrutinib reduced tumor-derived cytokines IL-6 and IL-10, which are negative regulators of NK cells. IFN-gamma secreted by nontumor cells was increased in the sera of CB17.SCID but not in NSG mice after ibrutinib treatment, providing further evidence of the increased NK function with ibrutinib. The detailed mechanisms of modulation of NK cell function by ibrutinib is currently under investigation. Conclusions: We report herein that ibrutinib enhanced NK cell-mediated cytotoxicity in mouse models of B-cell lymphomas. In addition to its direct effect on BTK inhibition in tumor cells, these data provide further evidence of the immune modulation function of ibrutinib. The role of ibrutinib in NK cell activation as observed in the current study may further expand the potential application of ibrutinib therapy. Disclosures Kuo: Pharmacyclics, LLC, an AbbVie Company: Employment, Other: Travel, Accommodations, and Expenses, Patents & Royalties: Pharmacyclics, LLC, an AbbVie Company; AbbVie: Equity Ownership. Hsieh:Pharmacyclics, LLC, an AbbVie Company: Employment. Whang:Pharmacyclics, LLC, an AbbVie Company: Employment; AbbVie: Equity Ownership. Huang:Juno: Equity Ownership; Pharmacyclics, LLC, an AbbVie Company: Employment; Five Prime: Equity Ownership; Merrimack: Equity Ownership. Sirisawad:AbbVie: Equity Ownership; Pharmacyclics, LLC, an AbbVie Company: Employment. Chang:AbbVie: Equity Ownership; Pharmacyclics, LLC, an AbbVie Company: Employment, Patents & Royalties: Pharmacyclics, LLC, an AbbVie Company.

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α-Pinene Enhances the Anticancer Activity of Natural Killer Cells via ERK/AKT Pathway

International Journal of Molecular Sciences ◽

10.3390/ijms22020656 ◽

2021 ◽

Vol 22 (2) ◽

pp. 656

Author(s):

Hantae Jo ◽

Byungsun Cha ◽

Haneul Kim ◽

Sofia Brito ◽

Byeong Mun Kwak ◽

...

Keyword(s):

Nk Cells ◽

Cancer Cells ◽

Natural Killer ◽

Nk Cell ◽

Granzyme B ◽

Akt Pathway ◽

Cell Cytotoxicity ◽

Anticancer Effects ◽

Nk Cell Cytotoxicity

Natural killer (NK) cells are lymphocytes that can directly destroy cancer cells. When NK cells are activated, CD56 and CD107a markers are able to recognize cancer cells and release perforin and granzyme B proteins that induce apoptosis in the targeted cells. In this study, we focused on the role of phytoncides in activating NK cells and promoting anticancer effects. We tested the effects of several phytoncide compounds on NK-92mi cells and demonstrated that α-pinene treatment exhibited higher anticancer effects, as observed by the increased levels of perforin, granzyme B, CD56 and CD107a. Furthermore, α-pinene treatment in NK-92mi cells increased NK cell cytotoxicity in two different cell lines, and immunoblot assays revealed that the ERK/AKT pathway is involved in NK cell cytotoxicity in response to phytoncides. Furthermore, CT-26 colon cancer cells were allografted subcutaneously into BALB/c mice, and α-pinene treatment then inhibited allografted tumor growth. Our findings demonstrate that α-pinene activates NK cells and increases NK cell cytotoxicity, suggesting it is a potential compound for cancer immunotherapy.

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