Teilah Kathryn Huth

Role of adaptive and innate immune cells in chronic fatigue syndrome/myalgic encephalomyelitis

Perturbations in immune processes are a hallmark of a number of autoimmune and inflammatory disorders. Chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) is an inflammatory disorder with possible autoimmune correlates, characterized by reduced NK cell activity, elevations in regulatory T cells (Tregs) and dysregulation in cytokine levels. The purpose of this article is to examine innate and adaptive immune cell phenotypes and functional characteristics that have not been previously examined in CFS/ME patients. Thirty patients with CFS/ME and 25 non-fatigued controls were recruited for this study. Whole blood samples were collected from all participants for the assessment of cell phenotypes, functional properties, receptors, adhesion molecules, antigens and intracellular proteins using flow cytometric protocols. The cells investigated included NK cells, dendritic cells, neutrophils, B cells, T cells, γδT cells and Tregs. Significant changes were observed in B-cell subsets, Tregs, CD4(+)CD73(+)CD39(+) T cells, cytotoxic activity, granzyme B, neutrophil antigens, TNF-α and IFN-γ in the CFS/ME patients in comparison with the non-fatigued controls. Alterations in B cells, Tregs, NK cells and neutrophils suggest significant impairments in immune regulation in CFS/ME and these may have similarities to a number of autoimmune disorders.

Characterisation of cell functions and receptors in Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME)

BackgroundAbnormal immune function is often an underlying component of illness pathophysiology and symptom presentation. Functional and phenotypic immune-related alterations may play a role in the obscure pathomechanism of Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME). The objective of this study was to investigate the functional ability of innate and adaptive immune cells in moderate and severe CFS/ME patients. The 1994 Fukuda criteria for CFS/ME were used to define CFS/ME patients. CFS/ME participants were grouped based on illness severity with 15 moderately affected (moderate) and 12 severely affected (severe) CFS/ME patients who were age and sex matched with 18 healthy controls. Flow cytometric protocols were used for immunological analysis of dendritic cells, monocytes and neutrophil function as well as measures of lytic proteins and T, natural killer (NK) and B cell receptors.ResultsCFS/ME patients exhibited alterations in NK receptors and adhesion markers and receptors on CD4+T and CD8+T cells. Moderate CFS/ME patients had increased CD8+ CD45RA effector memory T cells, SLAM expression on NK cells, KIR2DL5+ on CD4+T cells and BTLA4+ on CD4+T central memory cells. Moderate CFS/ME patients also had reduced CD8+T central memory LFA-1, total CD8+T KLRG1, naïve CD4+T KLRG1 and CD56dimCD16− NK cell CD2+ and CD18+CD2+. Severe CFS/ME patients had increased CD18+CD11c− in the CD56dimCD16− NK cell phenotype and reduced NKp46 in CD56brightCD16dim NK cells.ConclusionsThis research accentuated the presence of immunological abnormalities in CFS/ME and highlighted the importance of assessing functional parameters of both innate and adaptive immune systems in the illness.

Analysis of the Relationship between Immune Dysfunction and Symptom Severity in Patients with Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME)

Objective: Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME) is a disabling illness, characterised by persistent, debilitating fatigue and a multitude of symptoms. Immunological alterations are prominent in CFS/ME cases, however little is known about the relationship between CFS/ME severity and the extent of immunological dysfunction. The purpose of this study was to assess innate and adaptive immune cell phenotypes and function of two groups of CFS/ME patients, bedridden (severe) and mobile (moderate). Methods: CFS/ME participants were defined using the Centres for Disease Prevention and Control (1994 CDC) Criteria for CFS/ME. Participants were grouped into healthy controls (n=22, age=40.14 ± 2.38), moderate/ mobile (n=23; age=42.52 ± 2.63) and severe/bedridden (n=18; age=39.56 ± 1.51) CFS/ME patients. Flow cytometric protocols were used to examine neutrophil, monocyte, dendritic cells (DCs), iNKT, Treg, B, γδ and CD8+ T cell phenotypes, NK cytotoxic activity and receptors. Results: The present data found that CFS/ME patients demonstrated significant decreases in NK cytotoxic activity, transitional and regulatory B cells, γδ1T cells, KIR2DL1/DS1, CD94+ and KIR2DL2/L3. Significant increases in CD56-CD16+NKs, CD56dimCD16- and CD56brightCD16-/dim NKs, DCs, iNKT phenotypes, memory and naive B cells were also shown in CFS/ME participants. Severe CFS/ME patients demonstrated increased CD14-CD16+ DCs, memory and naive B cells, total iNKT, iNKT cell and NK phenotypes compared to moderate CFS/ME patients. Conclusion: This study is the first to determine alterations in NK, iNKT, B, DC and γδ T cell phenotypes in both moderate and severe CFS/ME patients. Immunological alterations are present in innate and adaptive immune cells and sometimes, immune deregulation appears worse in CFS/ME patients with more severe symptoms. It may be appropriate for CFS/ME patient severity subgroups to be distinguished in both clinical and research settings to extricate further immunological pathologies that may not have been previously reported.

A comparison of health status in patients meeting alternative definitions for chronic fatigue syndrome/myalgic encephalomyelitis

BackgroundSeveral diagnostic definitions are available for Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME) that varies significantly in their symptom criteria. This pilot study was conducted to determine whether simple biological and clinical measures differed between CFS/ME patients meeting the 1994 Centres for Disease Control and Prevention (CDC) criteria, the International Consensus Criteria (ICC), as well as healthy controls.MethodsA total of 45 CFS/ME patients and 30 healthy controls from the South East Queensland region of Australia provided a blood sample, reported on their current symptoms, as well as aspects of their physical and social health using the Short-Form Health Survey (SF-36), and the World Health Organisation Disability Adjustment Schedule 2.0 (WHO DAS 2.0). Differences were examined using independent sample t-testing.ResultsPatients fulfilling the ICC definition reported significantly lower scores (p < 0.05) for physical functioning, physical role, bodily pain, and social functioning than those that only fulfilled the 1994 CDC definition. ICC patients reported significantly greater (p < 0.05) disability across all domains of the WHO DAS 2.0.ConclusionsThese preliminary findings suggest that the ICC identifies a distinct subgroup found within patients complying with the 1994 CDC definition, with more severe impairment to their physical and social functioning.

Immune Abnormalities in Patients Meeting New Diagnostic Criteria for Chronic Fatigue Syndrome/Myalgic Encephalomyelitis

Background: Immunological abnormalities have been identified in Chronic Fatigue Syndrome/Myalgic Encephalomyelitis patients fulfilling the 1994 Centers for Disease Control diagnostic criteria. Significant developments have been made to diagnostic criteria, but potential immunological markers have not been assessed in patients fulfilling these latest clinical requirements. Therefore, this study evaluated immunological parameters in patients that also fulfill the latest diagnostic criteria available known as the International Consensus Criteria. Methods: The Immunological investigations including Natural Killer cell activity and phenotyping studies for dendritic cells, neutrophils, B cells and regulatory T cells were performed on whole blood samples collected from all participants using flow cytometric protocols. The physical functioning of all participants was also evaluated using scores from the Short Form Health Survey, and the World Health Organization Disability Adjustment Schedule. Results were compared according 1994 Centers of Disease Control and Prevention defined patients, and International Consensus Criteria defined patients, and healthy controls. Results: Natural killer cell activity was consistently and significantly decreased, and regulatory T cells were significantly increased in both patient groups compared to healthy controls. Differences were found in human neutraphil antigens and expression of natural killer cell receptors between patient groups. Highly significant correlations were also found between physical status and some immune parameters in International Consensus Criteria defined patients. Conclusion: This preliminary investigation on different diagnostic criteria suggests that the International Consensus Criteria may be more effective a detecting salient differences in the immune system.

Heat shock proteins and regulatory T cells.

Heat shock proteins (HSPs) are important molecules required for ideal protein function. Extensive research on the functional properties of HSPs indicates that HSPs may be implicated in a wide range of physiological functions including immune function. In the immune system, HSPs are involved in cell proliferation, differentiation, cytokine release, and apoptosis. Therefore, the ability of the immune system, in particular immune cells, to function optimally and in unison with other physiological systems is in part dependent on signaling transduction processes, including bidirectional communication with HSPs. Regulatory T cells (Tregs) are important T cells with suppressive functions and impairments in their function have been associated with a number of autoimmune disorders. The purpose of this paper is to examine the relationship between HSPs and Tregs. The interrelationship between cells and proteins may be important in cellular functions necessary for cell survival and expansion during diseased state.

Natural killer cells and single nucleotide polymorphisms of specific ion channels and receptor genes in myalgic encephalomyelitis/chronic fatigue syndrome

Aim The aim of this paper was to determine natural killer (NK) cytotoxic activity and if single nucleotide polymorphisms (SNPs) and genotypes in transient receptor potential (TRP) ion channels and acetylcholine receptors (AChRs) were present in isolated NK cells from previously identified myalgic encephalomyelitis (ME)/chronic fatigue syndrome (CFS) patients. Subjects and methods A total of 39 ME/CFS patients (51.69±2 years old) and 30 unfatigued controls (47.60±2.39 years old) were included in this study. Patients were defined according to the 1994 Centers for Disease Control and Prevention criteria. Flow cytometry protocols were used to examine NK cytotoxic activity. A total of 678 SNPs from isolated NK cells were examined for 21 mammalian TRP ion channel genes and for nine mammalian AChR genes via the Agena Bioscience iPlex Gold assay. SNP association and genotype was determined using analysis of variance and Plink software. Results ME/CFS patients had a significant reduction in NK percentage lysis of target cells (17%±4.68%) compared with the unfatigued control group (31%±6.78%). Of the 678 SNPs examined, eleven SNPs for TRP ion channel genes (TRPC4, TRPC2, TRPM3, and TRPM8) were identified in the ME/CFS group. Five of these SNPs were associated with TRPM3, while the remainder were associated with TRPM8, TRPC2, and TRPC4 (P<0.05). Fourteen SNPs were associated with nicotinic and muscarinic AChR genes: six with CHRNA3, while the remainder were associated with CHRNA2, CHRNB4, CHRNA5, and CHRNE (P<0.05). There were sixteen genotypes identified from SNPs in TRP ion channels and AChRs for TRPM3 (n=5), TRPM8 (n=2), TRPC4 (n=3), TRPC2 (n=1), CHRNE (n=1), CHRNA2 (n=2), CHRNA3 (n=1), and CHRNB4 (n=1) (P<0.05). Conclusion We identified a number of SNPs and genotypes for TRP ion channels and AChRs from isolated NK cells in patients with ME/CFS, suggesting these SNPs and genotypes may be involved in changes in NK cell function and the development of ME/CFS pathology. These anomalies suggest a role for dysregulation of Ca2+ in AChR and TRP ion channel signaling in the pathomechanism of ME/CFS.

Serum Immune Proteins in Moderate and Severe Chronic Fatigue Syndrome/Myalgic Encephalomyelitis Patients

Immunological dysregulation is present in Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME), with recent studies also highlighting the importance of examining symptom severity. This research addressed this relationship between CFS/ME severity subgroups, assessing serum immunoglobulins and serum cytokines in severe and moderate CFS/ME patients. Participants included healthy controls (n= 22), moderately (n = 22) and severely (n=19) affected CFS/ME patients. The 1994 Fukuda Criteria defined CFS/ME and severity scales confirmed mobile and housebound CFS/ME patients as moderate and severe respectively. IL-1β was significantly reduced in severe compared with moderate CFS/ME patients. IL-6 was significantly decreased in moderate CFS/ME patients compared with healthy controls and severe CFS/ME patients. RANTES was significantly increased in moderate CFS/ME patients compared to severe CFS/ME patients. Serum IL-7 and IL-8 were significantly higher in the severe CFS/ME group compared with healthy controls and moderate CFS/ME patients. IFN-γ was significantly increased in severe CFS/ME patients compared with moderately affected patients. This was the first study to show cytokine variation in moderate and severe CFS/ME patients, with significant differences shown between CFS/ME symptom severity groups. This research suggests that distinguishing severity subgroups in CFS/ME research settings may allow for a more stringent analysis of the heterogeneous and otherwise inconsistent illness.

Pilot Study of Natural Killer Cells in Chronic Fatigue Syndrome/Myalgic Encephalomyelitis and Multiple Sclerosis.

Patients with chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) and multiple sclerosis (MS) suffer from debilitating fatigue which is not alleviated by rest. In addition to the fatigue‐related symptoms suffered by patients with CFS/ME and MS, dysfunction of the immune system and, in particular, reduced natural killer (NK) cell cytotoxic activity has also been reported in CFS/ME and MS. The purpose of this pilot study was to compare NK cellular mechanisms in patients with CFS/ME and MS to investigate potential dysfunctions in the NK cell activity pathway. Flow cytometry protocols assessed CD56dimCD16+ and CD56brightCD16+/− NK cell expression of adhesion molecules, NK activating and inhibiting receptors, NK cell maturation and lytic proteins. All participants in this study were female and included 14 patients with CFS/ME, nine patients with MS and 19 non‐fatigued controls. The patient groups and the non‐fatigued controls were not taking any immunosuppressive or immune‐enhancing medications. In the MS cohort, KIR2DL5 was significantly increased on CD56brightCD16+/− NK cells and expression of CD94 was significantly increased on CD56dimCD16+ NK cells in comparison with the controls. Co‐expression of CD57 and perforin was significantly increased on CD56dimCD16+ NK cells from patients with CFS/ME compared to the MS and non‐fatigued control participants. The results from this pilot study suggest that NK cells from patients with CFS/ME and MS may have undergone increased differentiation in response to external stimuli which may affect different mechanisms in the NK cell cytotoxic activity pathway.

Characterization of Natural Killer Cell Phenotypes in Chronic Fatigue Syndrome/ Myalgic Encephalomyelitis

Objective: Natural Killer (NK) cells are classified into different phenotypes according to the expression of the surface markers CD56 and CD16. Each NK cell phenotype has a role in the immune response through cytotoxic activity or cytokine production. Reduced NK cell cytotoxic activity is a consistent finding in patients with Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME) and investigations into the potential causes of reduced NK cell cytotoxic activity have predominantly focused on total NK cells. The purpose of this study was to investigate and characterize four NK cell phenotypes in CFS/ME. Methods: Twenty nine CFS/ME patients (mean age ± SEM=48.28 ± 2.63) meeting the 1994 Fukuda definition and 27 healthy controls (mean age ± SEM=49.15 ± 2.51) were included in this study. Flow cytometric protocols identified CD56brightCD16-/dim, CD56dimCD16-, CD56dimCD16+ or CD56-CD16+ NK cells for the measurement of surface markers including adhesion molecules CD2, CD18, CD11a, CD11b and CD11c, natural cytotoxicity receptors, Killer Immunoglobulin Like Receptors, signalling lymphocytic activation molecules and cell maturation (CD57). Following stimulation, NK cell phenotype expression of CD107a and CD107b was measured as a marker for degranulation. Intracellular staining measured lytic proteins including perforin, Granzyme A and Granzyme B in the four NK cell phenotypes. Results: In the CFS/ME group, CD56brightCD16-/dim NK cell co-expression of adhesion molecules CD2 and CD18 was significantly reduced. Granzyme B was significantly decreased in CD56dimCD16+ and CD56-CD16+ NK cells from CFS/ME patients. CD57 expression on CD56dimCD16+ NK cells from CFS/ME patients was significantly increased. Conclusion: This is the first study to characterize four NK cell phenotypes in CFS/ME by investigating surface and intracellular molecules necessary for NK cell effector function. The data suggests that a combination of impairments in CD56dimCD16+ NK cells from CFS/ME patients may contribute to reduced cytotoxic activity of this phenotype.