Background
The innate immune system exhibits rapid effector functions and acts as the first line of defence, protecting the host cells while activating the adaptive immune system. Natural killer (NK) cells in particular are an important interface for the innate and adaptive immune systems; hence, impaired function potentially leads to immunological disturbances. The presence of immunological dysfunction may impair physiological functioning and may play a role in disease pathogenesis [
1]. NK cell dysregulation has been demonstrated in a number of illnesses, including human immunodeficiency syndrome (HIV), systemic lupus erythematosus (SLE), multiple sclerosis (MS) and Major Depressive Disorder (MDD) [
2-
4].
Patients with Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME) exhibit reduced NK and CD8
+T cell cytotoxic activity and differences in a number of adaptive immune cell phenotypes [
5,
6]. Significant decreases in NK cell cytotoxic activity in CFS/ME patients who were moderately affected by symptoms and characterised using the Fukuda criteria for the illness have been extensively reported [
5,
7-
23]. The reduced NK cell cytotoxic activity in CFS/ME patients may be associated with abnormalities in NK cell phenotypes, receptors or lytic proteins. Of the previous CFS/ME studies, five have also found significant differences in perforin and granzymes in NK cells of CFS/ME patients [
10,
20,
24-
26]. These changes in functional and phenotypic components of immune cells may be playing a role in the pathomechanism of CFS/ME.
CFS/ME is an enigmatic illness which has no known pathomechanism or cause, with diagnoses based on symptom specific criteria and a range of exclusions. Due to the multifactorial and complex nature of CFS/ME, there are often misconceptions and inconsistencies surrounding diagnosis which highlights the importance of thorough screening of participants in both clinical and research settings. This is particularly important in determining those with other illnesses such as major depression who may satisfy the CFS/ME symptom specific criteria and also demonstrate NK cell abnormalities [
2]. CFS/ME is characterised by persistent fatigue and a combination of symptoms which are often severely debilitating and the illness also tends to vary greatly in the nature of onset and symptom severity [
5,
7,
27-
31]. Moderate patients are mostly able to maintain normal daily activities but may be hampered by reduced mobility while severely affected CFS/ME patients experience high levels of daily fatigue and are typically housebound [
32].
Importantly, severe CFS/ME patients’ NK cell cytotoxic activity has only been reported in three previous investigations [
5,
7,
13]. Severely affected CFS/ME patients have demonstrated significant reductions in NK cell cytotoxic activity as well as increased NK cell receptor KIR3DL1 and enhanced plasma interleukin (IL)-4, tumour necrosis factor (TNF)-α and interferon (IFN)-γ [
7,
13]. This research also suggested a correlation between low NK cell cytotoxic activity and severity of CFS/ME, based on clinical status [
13]. As studies have investigated innate and adaptive immune cells in CFS/ME patients, it appears important to further examine functional parameters such as cell activity, receptors and adhesion molecules. Along with NK cell and CD8
+T cell aberrations [
5,
7,
11,
19,
33], dendritic cell (DC) phenotypes have been previously abnormal in CFS/ME patients [
5] although to date no study has assessed DC activity in the illness. Similarly, iNKT cells are seldom examined although one study found differences in iNKT cell phenotypes in CFS/ME patients [
5], suggesting the possibility of iNKT cell dysfunction in the illness and the need to assess cytotoxic granules in these cells. Cytotoxic granules have not previously been assessed in gamma delta (γδ) or regulatory T cells (Tregs) in CFS/ME patients, which may be cells of interest according to differences found in these cell types in previous research [
10,
16,
17,
34].
Previous research has outlined the presence of NK cell dysfunction and immunological abnormalities in CFS/ME patients although most studies only assessed moderately affected CFS/ME patients. Immune dysregulation can also be associated with the clinical features and aetiology of CFS/ME. This is supported as research has found significant clinical improvements in CFS/ME patients’ symptoms after B cell depletion [
35]. It is also possible that further immune cells may be affected by the illness and have yet to be assessed in CFS/ME patients. The current investigation was the first to measure the functional activity of DCs, neutrophils and monocytes, lytic proteins in iNKT, γδ and Tregs as well as receptors and adhesion molecules of NK, T and B cells in moderate and severe CFS/ME patients.
Discussion
This study is the first to provide an overview of cell function and receptor interactions, including assessing DC, neutrophil and monocyte function and receptors on T cells, BCRs and Bregs in moderate and severe CFS/ME patients. This is also the first study to examine lytic proteins in γδT cells, iNKT cells and Tregs in CFS/ME patients. Our results suggest that in some cases, functional immunological impairment may be related to differences in severity of CFS/ME patients, highlighting the variation in the illness. Significant alterations shown in moderate CFS/ME patients were often not present in severe CFS/ME patients and controls, revealing the possibility that moderate CFS/ME patients may have a different aetiology compared with the severely affected subgroup of CFS/ME patients.
Increased SLAM expression on total NK cells in moderate CFS/ME patients in this study may be a mechanistic response to the typically reduced NK cell cytotoxic activity in CFS/ME [
10,
11,
36]. SLAM is a receptor expressed on the surface of T, B, NK and DC cells, functioning as an activating adaptor protein to amplify the recruitment of inflammatory cells, such as DCs, by activating IFN-γ [
37-
39]. SLAM receptors regulate NK cell activity via association with SLAM associated protein (SAP) family adapters, where binding receptors are then coupled to the Src kinase FynT to evoke protein tyrosine phosphorylation signals [
40]. Heightened expression of SLAM in moderate CFS/ME may enhance the ability of NK cells to undergo cytotoxic activities [
5,
7-
11]. The activating receptor NKp46 is also typically involved in the recognition and lysis of target cells and was reduced in CD56
brightCD16
dim NK cells of severe CFS/ME patients [
41]. NKp46 is highly expressed on the CD56
brightCD16
dim NK cell subset and although it is only weakly involved in cytotoxic activation, this reduction may contribute to the reduced NK cell cytotoxic activity prevalent in severe CFS/ME patients [
5,
41,
42]. The expression of SLAM and NKp46 on NK cells significantly differed between moderate and severe CFS/ME patients, suggesting that perhaps these severity subgroups may vary in immunological presentation, as proposed by previous studies [
5,
7,
13].
Effector memory and CD45RA effector memory cells demonstrate NK-like functions as they have the ability to detect abnormal major histocompatibility complex (MHC) expression and have a high capability for cytotoxic activities [
43]. Our findings suggest that the number of CD45RA effector memory CD8
+T cells of moderate CFS/ME patients may be enhanced due to sub-optimal function. In T cells, CD45RA effector memory cells are upregulated following cytokine directed proliferation, indicating that the subset is generated via homeostasis rather than antigen-dependent pathways [
44,
45]. Moderate CFS/ME patients have an increased number of CD8
+T CD45RA effector memory cells, potentially as a result of homeostasis where the cells may not be effectively undergoing degranulation and apoptosis [
44,
45].
This research also found reductions in KLRG1 expression in total CD8
+T and naïve CD4
+T cells of moderate CFS/ME patients, which suggests that these cells may have a reduced ability to inhibit T cell function and activation. KLRG1 ligation inhibits the nuclear factor of activated T cells (NFAT) signalling pathway and downregulates CD95 mediated lysis to inhibit the activation of T cells [
46]. Hence, blockades of inhibitory receptors tend to improve CD8
+T cell responses by preventing inhibitory pathways [
47]. It is therefore possible that reduced KLRG1 may be contributing to the pro-inflammatory response and T cell activation often found in CFS/ME patients [
10,
48].
Increased KIR2DL5 on CD4
+T cells in moderate CFS/ME patients may also be associated with alterations in KIR receptors in T cells in the same cohort [
5,
7]. KIR2DL5 is an inhibitory KIR found in variable proportions of circulating T cells [
49,
50] which is directly linked to a greater number of random combinations of KIR receptors expressed on these cells, which may be influencing optimal T cell functions in the illness [
51]. Enhanced inhibitory signalling and modulation of immune responses are typical attributes of increased BTLA expression in T cells which may be present in moderate CFS/ME patients who have amplified expression of inhibitory receptor BTLA4 in central memory CD4
+T cells [
52]. Activation and function of CD4
+T cells by NK cells is dependent on the engagement of the β
2 integrin LFA-1. LFA-1 adhesion is necessary for optimal cytotoxic activity by both NK cells and CD8
+T cells, also mediating NK cell degranulation via synergy with NKG2D [
53]. Decreased expression of LFA-1 on central memory CD8
+T cells in moderate CFS/ME patients suggests that there may be a lack of LFA-1 adhesion in CFS/ME which is required for ideal cytotoxic activity by NK cells and CD8
+T cells [
53]. Similar to the pattern shown in SLAM and NKp46 receptors on NK cells, CD45RA effector memory CD8
+T cells and CD4
+T and CD8
+T cell receptors significantly differed between moderate and severe CFS/ME patients.
Cellular adhesion may be important in CFS/ME as it is required for target cell contact and NK cell effector function. Regulation of adhesion molecules is necessary for integrin target cell ligand interactions as the release of adherence results in lymphocyte movement [
54]. CD2 expression in CD56
dimCD16
− NK cells is reduced in moderate CFS/ME patients compared with severely affected patients, suggesting that these cells may have an impaired ability to adhere to target cells. This confirmed previous findings where CD2/CD18 co-expression was reduced in the same CD56
dimCD16
− NK cell phenotype in a cohort of moderate CFS/ME patients [
24]. Increased CD2 is often associated with a higher cytotoxic ability [
55] as CD2 acts as a contributor to induce NK cell activation [
54,
55]. Higher expression of CD2 in severe CFS/ME patients potentially implies that more NK cells in these patients are in an active state and may have a greater ability than the moderate CFS/ME patients to induce NK cell activation and cytotoxic activities [
55]. CD18
+/CD2
− CD56
dimCD16
− NK cells were also increased in the moderate CFS/ME patients, strengthening the theory that CFS/ME patients may have a weakened ability to activate NK cells as well as having impaired NK cell cytotoxic activity. Adhesion molecules CD18 and CD2 on CD56
dimCD16
− NK cells were also significantly altered in the moderate CFS/ME patient group compared with the severe CFS/ME patients, who appeared similar to the controls. In the case of CD18
+CD11c
− on the same CD56
dimCD16
− NK cell subset, however, increases in CD18
+CD11c
− increased in the moderate CFS/ME patients and significantly increased in the severe CFS/ME patients. Expression of the adhesion marker CD11c is heterogeneous and variable in NK cells although, typically activated NK cells are CD11c
+ [
55]. Increased CD18
+CD11c
− on CD56
dimCD16
− NK cells in severe CFS/ME patients indicates that these patients may have a reduced ability to adhere or that they may have a low number of activated NK cells. Therefore, differences in CD18
+CD11c
− adhesion molecules in severe CFS/ME patients may be associated with the reduced NK cell cytotoxic activity found in the illness [
5,
10,
11].
CFS/ME symptom severity and presentation may be related to the immune dysregulation shown as the immune system interacts with physiological functioning via a number of body systems, including the central nervous system, digestive system and endocrine system [
56]. Unrefreshing sleep and sleep disturbances are symptoms of CFS/ME and reports have indicated that NK cells are altered after sleep deprivation, demonstrating interactions between physiological symptoms and the immune system [
57], particularly in CFS/ME patients. Similarly, it has previously been suggested that clinical severity status appears to be associated with reduced NK cell activity in CFS/ME patients [
7,
13]. Although there are limited research findings for severe CFS/ME patients, the differences in NK cells, CD4
+T and CD8
+T cells between severity groups, found in this research, suggest that immune dysfunction in CFS/ME may be related to clinical symptoms and hence severity.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
SH participated in the study design, patient recruitment, laboratory analysis, statistical analysis and drafted the manuscript. EB, SJ, SR, DS and SMG participated in the study design, patient coordination, data interpretation and drafting of the manuscript. NW, TN and TH assisted in the collection and analysis of laboratory data and revised the manuscript. All authors read and approved the final manuscript.