Natural killer cells in patients with severe
chronic fatigue syndrome
E. W. Brenu, S. L. Hardcastle,
G. M. Atkinson, M. L. van Driel,
S. Kreijkamp-Kaspers, K. J. Ashton,
D. R. Staines & S. M. Marshall-Gradisnik
Autoimmunity Highlights
ISSN 2038-0305
Autoimmun Highlights
DOI 10.1007/s13317-013-0051-x
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DOI 10.1007/s13317-013-0051-x
REVIEW
Natural killer cells in patients with severe chronic fatigue
syndrome
E. W. Brenu • S. L. Hardcastle • G. M. Atkinson •
M. L. van Driel • S. Kreijkamp-Kaspers •
K. J. Ashton • D. R. Staines • S. M. Marshall-Gradisnik
Received: 28 November 2012 / Accepted: 6 March 2013
Ó Springer-Verlag Italia 2013
Abstract Maintenance of health and physiological
homeostasis is a synergistic process involving tight regulation
of proteins, transcription factors and other molecular processes. The immune system consists of innate and adaptive
immune cells that are required to sustain immunity. The
presence of pathogens and tumour cells activates innate
immune cells, in particular Natural Killer (NK) cells. Stochastic expression of NK receptors activates either inhibitory
or activating signals and results in cytokine production and
activation of pathways that result in apoptosis of target cells.
Thus, NK cells are a necessary component of the immunological process and aberrations in their functional processes,
Electronic supplementary material The online version of this
article (doi:10.1007/s13317-013-0051-x) contains supplementary
material, which is available to authorized users.
E. W. Brenu S. L. Hardcastle G. M. Atkinson
S. M. Marshall-Gradisnik
Griffith Health Institute, School of Medical Science, Griffith
University, Gold Coast, QLD, Australia
E. W. Brenu S. L. Hardcastle G. M. Atkinson
D. R. Staines S. M. Marshall-Gradisnik
The National Centre for Neuroimmunology and Emerging
Diseases, Griffith University, Gold Coast, QLD, Australia
E. W. Brenu (&)
Immunology Research Group, Centre for Medicine and Oral
Health, Griffith University, GH1, Room 7.59, Southport, QLD
4215, Australia
e-mail: e.brenu@griffith.edu.au
M. L. van Driel D. R. Staines
Queensland Health, Gold Coast Public Health Unit, Robina,
Gold Coast, QLD, Australia
S. Kreijkamp-Kaspers K. J. Ashton
Faculty of Health Sciences and Medicine, Bond University,
Robina, QLD, Australia
including equivocal levels of NK cells and cytotoxic activity
pre-empts recurrent viral infections, autoimmune diseases
and altered inflammatory responses. NK cells are implicated
in a number of diseases including chronic fatigue syndrome
(CFS). The purpose of this review is to highlight the different
profiles of NK cells reported in CFS patients and to determine
the extent of NK immune dysfunction in subtypes of CFS
patients based on severity in symptoms.
Keywords Chronic fatigue syndrome Natural killer
cells Cytotoxicity Perforin Granzymes
Introduction
Natural killer (NK) cells are granular lymphocytes originating from the CD34 hematopoietic progenitor cell lineage and are found in the peripheral blood, spleen, bone
marrow and lymph nodes. The composition of NK cells in
comparison to the total lymphocyte population is about
15 % [1]. These cells are important in the principal innate
immune defence in response to pathogen invasion following recognition. NK cells are imperative during viral and
microbial infection and tumour development, aiding in the
body’s immunity through cytokine secretion and cytotoxic
activity, which induces apoptosis in target cells. Thus, NK
cells are vital for pathogen clearance prior to the adaptive
immune response. Aberrant production of cytokines and
induction of cytotoxic activity are related to a number of
disease presentations such as rheumatoid arthritis [2],
chronic obstructive pulmonary disorder [3], neurological
conditions including Alzheimer’s and multiple sclerosis [4,
5] and cancers [6–9]. In particular, NK cell cytotoxic
dysfunction has been associated with chronic fatigue syndrome (CFS).
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Subsets of NK cells
Natural killer cells can be grouped into two types, however,
further classifications generates at least five subsets of NK
cells [10, 11]. These cells are mainly grouped according to
the presence of the Fc gamma receptor III (CD16) and the
neural cell adhesion molecule (CD56). The density of these
markers on the NK cells defines the classification of different
subsets of NK cells. Thus, NK cells can be grouped into
CD56brightCD16dim, CD56brightCD16-, CD56-CD16bright,
CD56dimCD16bright and CD56dimCD16-NK cells [10, 11].
About 90 % of the NK cells in the periphery are
CD56dimCD16bright while 10 % express CD56bright on their
cell surfaces [10]. NK cells with a high density of the CD16
molecules are considered highly cytotoxic and secrete low
levels of cytokines while those densely populated with CD56
markers are the dominant producers of NK-related cytokines
and are less cytotoxic [10]. The cytokines secreted by these
NK cells include granulocyte macrophage colony-stimulating factor (GMC-SF), IFN-c, TNF-a, IL-10 and IL-13. The
ability to induce cytotoxic activity and produce cytokines is
vital for sustained physiological homeostasis.
NK cell receptors
Natural killer cells express a number of activating and
inhibitory receptors generated from genes with variable or
conserved sequences. The most variable and polymorphic
NK receptors are the killer cell immunoglobin-like receptor
(KIR) family of receptors while the others such as NKG2D
are highly conserved [12]. KIRs are a family of inhibitory
receptors that reside as transmembrane proteins, with two
or more extracytoplasmic immunoglobin-like domains with
either short or long cytoplasmic tails that have an immunoreceptor tyrosine-based activation or inhibition motif
(ITAM and ITIM, respectively). KIRs contain a long
cytoplasmic tail and do not associate with adaptor molecules. The activation of KIRs is dependent upon the recognition of MHC class I molecules. The primary function
of these receptors is the inhibition of cytotoxic activity—
the inhibitory KIRs are associated with having a longer
cytoplasmic tail. However, some KIRs containing ITAM
motif, KIR2DS/3DS, are known to stimulate cytotoxic
activity [13].
Natural killer cells undergo cytotoxic activation by
exogenous activation of surface receptors. Activating NK
receptors including, NKp46, NKp30 and NKp44, all share
the association of a signalling peptide existing on cytoplasmic tail, known as ITAM. ITAMs are highly conserved peptides containing tyrosine residues [14]. The
ITAM peptide is a crucial intermediate between activation
of the surface receptor and downstream effector signalling,
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which in the case of NK cell is cytotoxic activation and
the production of cytokines including IFN-c, tumour
necrosis factor b (TNF-b), IL-10 and IL-13. The activation
receptors of NK cells have a broad specificity and can be
activated by target cell antibody interaction, such as that
of an antigen presenting cell or B cell, or through the
recognition of the MHC class I-like complex. It has been
indicated that numerous receptor activation and cross
linking between these receptors is required for NK cytotoxic activation [15].
Natural killer cell activation predominantly relies on
receptors. Upon recognition of the adaptor molecule, the
ITAM components of the receptor are activated through the
phosphorylation of its tyrosine residues [14]. This temporary binding site now has an affinity to activate of ITAM,
which leads to the recruitment of a src-family kinase (SH2)
pathway and downstream syk/ZAP70 transcription. Syk
acts through the PI3K-dependent pathway to activate Rac1,
PAK1, MEK and ERK pathways increasing calcium entry,
degranulation, recruitment of perforin and granzyme contained in lytic granules and cytokine gene transcription [16,
17]. These activating receptor molecules on target cells
may be antibodies or MHC class 1-like molecules,
requiring activation of multiple receptors and receptor
cross-linking to activate cytotoxicity [18, 19]. Inhibition of
NK cells occurs in the absence of a structurally sufficient
MHC class I molecule, giving rise to the ‘missing self’
theory of cytotoxic inhibition [20]. Recognition of the
target cell MHC class I, prompts ITIM phosphorylation at
the tyrosine residues to recruit and activate SHP-1/2
phosphatases. SHP1/2 dephosphorylates activated ITAM
pathway constituents, Syk and ZAP70, thereby inhibiting
cytotoxic activation and cytokine production.
NK cell cytotoxic activity
Natural killer cells can induce apoptosis in target cells
through granule-mediated and non-granule-mediated pathways. Granule-dependent cytotoxic induction is the most
specialised cytotoxic function of the NK cell [21, 22].
However, the significance of non-granule-mediated pathways is evident in the diversity of lethality of the NK cell
including antibody-dependent cell-mediated cytotoxicity
[23–27], TRAIL and FasL death receptor pathways [28–
30].
The relevance of the cytolytic granule-mediated pathway to the CFS disease state is supported by a growing
body of evidence highlighting cytotoxic dysfunction and
the immune system [31–37], summarised by Bansal et al.
[38]. By activation of the granule-mediated pathway, NK
cells secrete perforin and granzymes into the target cells.
Perforin is a protein that either forms pores on the plasma
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Autoimmun Highlights
membrane of the target cells to facilitate the passage of
granzymes into the target cells or fragments the host cell
endosomes, which contain granzymes [21]. They may also
be directly involved in cytolytic activity and once in the
target cells, granzymes bind to cell organelles to activate
either caspase-dependent or -independent apoptosis. These
mechanisms include nuclear envelope disruption leading to
DNA degradation, disruption of mitochondrial transmembrane potential and independently activates Ape1-mediated
bcl-2 overexpression [39]. NK cells may also contain a
memory component that assists in future invasions by the
same antigen. The primary pro-apoptotic component of NK
cells—granzymes, are currently categorised into five types,
only three of which have directly known functions—
granzymes A, B and K [40].
Granzymes A and K have similar functions and are
known to activate slow apoptosis while granzyme B is
associated with the activation of rapid apoptosis [41].
Granzyme B induces a caspase-dependent mechanism of
apoptosis while granzyme A is caspase independent,
inducing cell death through single stranded DNA degradation, disrupting plasma membrane integrity and mitochondrial transmembrane potential [39, 42–44]. The exact
mechanism of perforin is still under investigation, however, mounting evidence tends to support oligomerisation
within the target cell membrane, leading to the formation
pore-like structures [45, 46]. These pores are then the
gateway for NK cell-derived granzymes to enter the target
cell and elicit their effect. NK cell cytotoxic activity also
occurs through a number of other secondary pathways
including IFN-c, TNF-a and Fas-ligand pathways, the
dysregulation of which may be involved in the mechanism
of CFS.
Chronic fatigue syndrome
Presently, an indefinable aetiology and mechanism of CFS
precludes effective diagnosis posing substantial anxiety
among patients and family members. CFS is a heterogeneous disorder with multi-factorial characteristics affecting physiological processes including, endocrine,
neurological, immunological and metabolic processes [47–
54]. Substantial physical and mental weaknesses are
associated with CFS including but not limited to severe
disabling fatigue, interruptions in sleep, headaches, swollen lymph nodes, cognitive disturbances and muscle pain
in the absence of swelling [55]. CFS is neither age- nor
gender-specific, however, females are more likely to be
affected than males [56–58]. CFS is an unexplained disorder with a prevalence of 0.4–1 % worldwide [59].
Nonetheless prevalence of CFS varies among patients
[60].
NK cells in CFS
Chronic fatigue syndrome is known to be associated with a
reduction in NK lytic activity and in some cases an irregular distribution in the levels of NK subtypes. In a range of
studies, NK cytotoxic activity has been measurably
decreased as compared to healthy controls [31, 33, 34, 61–
64]. There is no standard definition for CFS, however, a
number of criteria have been generated to assist physicians
in disaffecting CFS from other known and characterised
disorders [59]. Similarly, our longitudinal investigations of
CFS have shown that reduced cytotoxic activity in patients
with CFS is maintained during the course of the disease
and does not notably fluctuate or associate with seasonal
changes. Incidentally, we have recently studied a group of
severely bed-ridden patients, due to the symptoms of CFS.
In these individuals, we have shown that, similar to the
CFS patients who have some level of mobility outside the
home, these patients demonstrated a significant decrease in
cytotoxic activity in their NK cells. However, in previous
investigations, reductions in NK cell function have been
associated with reduced levels of NK CD56bright cells [34].
A similar trend was not observed in our severe bed-ridden
population and a study of the longitudinal expression of
these subsets demonstrated that these cells are not consistently reduced over time or during the course of the disease
[31]. This is consistent with the NK cells subset studies in
the literature where consistencies in the levels of NK cells
have not been observed across all studies. The heterogeneity of CFS may be associated with these findings; however, it posits that levels of NK cells may not be an
appropriate marker for identifying and distinguishing CFS
from the general population. The observation of reduced
NK cell cytotoxicity in both mobile and bed-ridden cases
of CFS is important to the current knowledge of the disease. In the severe cases of CFS, differences in the KIR
receptors may be associated with the disease presentation.
Notably the transcriptional levels of some KIRs are significantly decreased in the CFS patients compared to the
controls while the expression of KIR2DS5 is not observed
in all CFS patients [65].
Allotypic and haplotypic differences in the expression of
these KIRS may affect the induction of cytotoxic activity
[66, 67]. Due to the high polymorphic nature of KIR, it is
proposed that specific polymorphisms may be associated
with the differences in expression of CFS patients [68]—
either due to a pre-transcriptional or a compensatory
means. However, studies are yet to provide details on such
genomic data of KIRs in CFS patients, which may be
further complicated by the heterogeneity and ambiguity of
the disease presentation and symptoms. In the absence of
appropriate diagnostic tools and a well-characterised suite
of biomarkers, CFS remains complex. A unifying theme in
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the literature is the presence of dysfunctional immune cells
specifically NK cells. Most immunological studies in CFS
are concerned with the cytotoxic activity and phenotypic
distribution of these cells.
Equivocal levels of NK cells and phenotypes in CFS
Chronic fatigue syndrome studies associated with peripheral NK cell phenotypes or subsets are contradictory.
Studies have reported increases, decreases and no change
in peripheral distribution of NK cell phenotypes and
overall NK cells in comparison to non-fatigued participants. Regardless of these inconsistencies, alterations in
NK phenotypes have adverse consequences on immune
function owing to the cytokine secretion and cytolytic
properties of these cells. Characterisation of NK cells into
CD56bright and CD56dim permits the determination of the
distribution of cytokine producing and cytotoxic NK cells.
Overall NK cell numbers may be increased or decreased
in some CFS patients [69, 70]. Similarly, CD56bright NK
cells may be increased or decreased in some CFS patients
[34, 70]. The consequence of equivocal levels of CD56bright
NK cells in CFS patients is unknown. Nonetheless,
CD56bright NK cells are highly resistant to apoptosis and
therefore have in increased life span in comparison to the
CD56dim NK cells [71, 72]. CD56bright NK cells in close
relation to T cells may be increased following elevations in
their numbers perpetuating autoimmune responses [73].
The presence of heightened levels of CD56bright NK cells
may suggest the presence of inflammation in the periphery.
The diversity in chemokine receptor expression on the
subtypes of NK cells is related to their sites of manifestation during inflammation, hence, CD56bright NK cells
expressing CCR5 are present in inflamed areas with high
incidence of RANTES and MIP-1a and b [74, 75]. These
NK cells are substantially activated following from their
interactions with monocytes [76]. Interestingly, reduction
in the levels of CD56bright NK cells is suggestive of differentiation of the CD56bright NK cells into the CD56dim
NK cells [77]. Concurrent expression of high levels of
CD56dim NK cells and low level of CD56bright NK cells
may explain this phenomenon in CFS patients [70]. In
diseases like AIDS, reduction in the levels of CD56dim NK
cells correlates with decreases in NK cytotoxic activity
[78]. The equivocal levels of NK phenotypes limits the
acceptance of this assumption in CFS patients nonetheless,
it is possible to posit that in some CFS patients with
marked reduction in NK phenotypes and activity a similar
disease profile may be observed. The heterogeneity of CFS
may confuse these findings hence, levels of NK cells may
not be appropriate markers for identifying and distinguishing CFS from the general population.
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NK cytotoxic activity in CFS
Although NK cell phenotypes and overall NK cell numbers
in the periphery are unpredictable, consistent decreases in
the cytotoxic activity occur in most CFS patients [1, 5, 36,
64, 69, 79–83]. A rationale for decreases in cytotoxic
activity remains to be determined, however, these may be
associated with altered lytic proteins in particular perforin
and granzymes [37]. Lytic proteins are important factors in
the granule-dependent pathway of cytolysis. Perforin contains a membrane attack complex and a C2 domain that
contains Ca2? [84]. In CFS patients, perforin gene
expression may be increased in conjunction with relatively
low to normal levels of granzymes [32, 37, 85].
Perforin is an important indicator to cytotoxic activity as
it is an absolute necessity for granule-related apoptosis [21,
86]. Incidentally, mice lacking perforin demonstrate
reduced apoptosis [86, 87]. Trafficking or exportation of
granzymes into the target cell is dependent on the availability of perforin thus its deficiency pre-empts decreased
cytotoxic activity owing to the paucity in the available
granzymes to induce apoptosis [21, 45]. Granzyme distribution in some CFS patients may be reduced [32]. It is
known that during development the level of perforin in the
NK cell is related to the expression of CD56. Following
maturation, a substantial proportion of NK cell-related
perforin is detected in the CD56dim NK cells in comparison
to the CD56bright NK cells [88]. Upregulation of perforin is
regulated by important proteins including IFN-b, IL-2, IL6, IL-12, IL-15 and IL-21 [89, 90]. In CFS IL-2, IL-6, IL15 and IL-21 are known to be characterised by alterations
in cytokine levels, this may be a contributory factor to the
decrease in cytotoxicity [91, 92].
Granzyme decrease in CFS may be attributed to
decreases in perforin although correlations remain to be
proven. Granzymes are serine proteases, in humans they
include, granzyme A, B, H, M and K [93–96]. Granzyme A
and B are the most characterised and they induce apoptosis
via distraction of endoplasmic reticulum SET complex or
activating of caspase 3 following cleavage of substrates as
previously mentioned [97]. Granzymes are found in the
extracellular fluids such as plasma, cerebrospinal fluids and
synovial fluid and are therefore implicated in the regulation
of inflammation [98]. The diverse role of perforin and
granzymes in cell death-related pathways is paramount to
immune function during infections. Hence, in CFS recurring infections may occur as a consequence of aberrations
in cytotoxic activity. Importantly, NK cells employ a
number of other cytotoxic pathways that may require further investigations in CFS to ascertain the exact pathway(s) that have an involvement with reduced NK
cytotoxic activity.
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Other factors that may affect efficient cytotoxic activity
in NK cells of CFS patients may be related to cytokine
production and secretion by NK cells. Cytotoxic activity
may occur via IFN-c and TNF-a. Therefore, changes in
these cytokines in the CFS patients may also have a contributory role to the observed decreases in cytotoxic activity
in these patients. Incidentally, cytokine studies in CFS
although not NK cell-specific may have a role in reduced
lysis [99, 100]. Importantly, in some CFS patients, significant increases in IFN-c and TNF-a may indicate induction
of other cell death pathways [31]. Nonetheless, this does not
reflect an increase in cytotoxic activity as reduced cytotoxic
activity still persists even when these pro-inflammatory
cytokines are increased [31, 32]. IFN-c is the most abundant
cytokine secreted by NK cells, in particular CD56bright NK
cells. Longitudinal assessments of cytokines in CFS
patients explicitly illustrate nonconformities in the presentation of cytokine production over the cause of CFS [31].
Shifts towards pro-inflammatory cytokines such as IFN-c
and TNF-a may occur initially but dissipate in time. In most
cases as we have observed in our CFS patients, an increase
in IFN-c and TNF-a occurred in coincidence with an
increase in anti-inflammatory cytokine IL-10.
NK receptors in CFS
Natural killer receptors are the least investigated NKrelated parameters in CFS. Currently, only one study has
examined the relationship between NK receptor expression
in CFS patients [65]. NK cells expression a varying number
of activating and inhibitory receptors that may have stochastic presentations. It is possible to posit that failures in
the regulation of the expression of these receptors can
affect NK function during viral invasion. For example,
elevated levels of inhibitory KIRs such as KIR3DL1 may
result in decreased NK cell lyis in patients with lung cancer
[101]. KIR receptors are exceedingly polymorphic and
KIR3DL1 is no exception as it expresses eight different
KIR3DL1 allotypes with differing sensitivities to antigen
binding [102–105]. HIV and spondyloarthritis patients
demonstrate high levels of KIR3DL1 [13, 106–108].
Similarly, the incidence of KIR3DS1 in some CFS
patients exceeds that of non-fatigued controls [65]. A
similar gene encodes KIR3DL1 and KIR3DS1 [109],
suggesting a potential link between these receptors in CFS
and cytotoxic activity. Certain ligands of these receptors
may also be elevated in CFS patients [65]. Diversity in the
KIR receptor polymorphism may generate receptors with
differing haplotypes that are specific to CFS. Further
studies are required to provide extensive details into the
polymorphisms of KIRs in CFS patients, which may be
further complicated by the heterogeneity and ambiguity of
the disease presentation and symptoms.
NK gene expression studies in CFS
Natural killer gene expression or molecular studies in CFS
are lacking, currently, only two studies have investigated
mRNA and microRNA (miRNA) studies in CFS. Perhaps
the lack of such studies relates to the cost associated and
volume of blood required for the preferential isolation of
NK cells. In PBMCs, expression of GZMA and GZMB is
reduced in CFS patients in comparison to non-fatigued
controls. GZMA and GZMB are genes for the protein
granzyme A and B, respectively. These reductions may
correlate with the protein production. In our previous
studies, preferential examination of lytic protein genes in
CFS patients revealed a significant expression in the perforin gene PRF1 while GZMA and GZMK were significantly reduced in the CFS patients [32]. The exact cause of
increase in the expression of perforin is not known, however, as perforin proteins were not measured in these CFS
patients, it is difficult to predetermine an association
between these PRF1 expression and perforin protein.
MicroRNAs are non-coding small RNA molecules with
regulatory roles in the expression of genes including
translation repression or mRNA degradation [110, 111]. In
CFS, NK cell expression of miR-10a, miR-21, miR-103,
miR-106, miR-146a, miR-150, miR-17-5p, miR-191 and
miR-223 are down-regulated in comparison to non-fatigued
controls [112]. Most of these miRNAs have been linked to
a number of cancers. An association or the role of these
miRNAs in NK cell-related activities is yet to be determined nonetheless these miRNAs are attributed to a
number of diseases and physiological processes. Most of
these miRNAs are associated with the presentation of a
number of different cancers and are involved in apoptosis,
cell proliferation and development. Importantly, miR-21,
miR-150 are implicated in the development of lymphocytes
and thus they may have similar effects in NK cells [113,
114]. Decreases in the expression of miR-10a occur in
chronic myeloid leukaemia [115]. MiR-10a preserves
vascular integrity by targeting HOXA1, MAP3K7 and
bTRC [116]. MiR-146a upon induction has been shown to
target TNF receptor-associated factor 6 (TRAF6) and the
IL-1 receptor associate kinase 1 (IRAK1) genes, and these
are important in the regulation of TLRs and inflammation
[117]. In many cancers the presence of miR-146a resulted
in cell proliferation [118]. Bacterial antigens and proinflammatory cytokines stimulate the expression of miR146a, which in turn may suppress the secretion of inflammatory cytokines [119]. Similarly, miR-21 promotes
tumour growth owing to its oncogenic properties and its
role in inflammation and T cell-related activities [120].
These studies on miRNAs have elucidated an important
role of miRNAs in NK cells, as they regulate the expression of immune-related genes. However, these studies are
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limited as they have not identified the exact miRNA target
genes in CFS patients. Such studies may be instrumental in
unexplained disorders such as CFS, further research, may
be required to establish these links.
NK cytotoxic activity in severe CFS patients
Cytotoxic activity of the NK cells was measured by the
ability of the cells to lyse the K562 effector cells. The NK
cytotoxic activity against K562 cells was significantly
decreased in the MCFS and the SCFS group (P \ 0.05)
compared to the non-fatigued control group (Fig. 1).
Multiple comparison tests revealed significant decreases
between the SCFS patients and the control group only.
There were no significant differences between the MCFS
and the control group or the SCFS and the MCFS group.
*
% Ly si s
30
20
10
0
SCFS
MCFS
Control
Fig. 1 Decreased NK lysis in MCFS and SCFS group compared to a
non-fatigued control group. The percent lysis of NK cells in each
group is represented above where the white bar represents the results
from the non-fatigued control group and the black bar represents the
SCFS group. Asterisk denotes statistical significance where P \ 0.05
and data is represented as the mean ± SEM
25
*
20
15
(%)
The results from studies on NK cells in CFS patients
suggest a potential mechanism of CFS can be identified
through a thorough study of NK cell-related activities.
From our observations, reductions in lytic proteins, genes
and further decreases in miRNA genes [31, 34, 112],
cumulatively affect efficient cytotoxic activity in CFS
patients. Similarly, the polymorphic alleles of the KIR
receptors may not allow efficient pathogenic and antigenic
targeting of the NK cell, as an overabundance in the
inhibitory KIRs may abort or impede cytotoxic activity
[121]. The extent immune dysfunction in subtypes of CFS
patients may differ among subgroups of patients. CFS
patients may have variations in the severity of their
symptoms, for example a distinct subgroup of patients
maybe housebound as they suffer from high levels of
fatigue and CFS-related symptoms compared to other
sedentary CFS patients [122]. Their severe persistent and
incapacitating symptoms probably exclude these patients
from CFS-related studies. Hence, we examined for the first
time NK cell-related parameters including cytotoxic
activity, phenotypes and KIR receptor expression in
patients with severe CFS (SCFS) in comparison to sedentary or moderate CFS (MCFS) patients and non-fatigued
controls. Currently, these studies have not been performed
in this group of CFS patients.
*
40
Expression of Receptor on Cell Surface
Implications for severe CFS patients
50
10
5
0
SCFS
MCFS
Control
Fig. 2 Expression of KIR3DL1 in SCFS, MCFS and a non-fatigued
control group. The above bar graph is based on the flow cytometric
analysis of KIR3DL1. This was the only receptor that was significantly (P \ 0.05) increased in the MCFS group in comparison to the
SCFS and non-fatigued controls. There was no significant difference
between the SCFS group and the MCFS group. Asterisk denotes
statistical significance where P \ 0.05 and data is represented as the
mean ± SEM
expression was significantly different between the MCFS
group and the non-fatigued controls. There was a general
trend of reduced receptor expression in the non-fatigued
controls in comparison to the other two groups. However,
most of these observations were not statistically significant
(data not shown).
NK receptors in severe CFS patients
NK cytokines in severe CFS patients
The percentage of NK receptor expression was determined
following preferential gating on isolated NK cells in a
forward and side scatter plot. This was then extrapolated on
to six plots for CD56 versus the six NK receptors assessed.
Significant changes in NK receptors were observed in only
one receptor, KIR3DL1 (CD158e) (Fig. 2). KIR3DL1
123
In the present study, plasma cytokines were investigated in
SCFS, MCFS and non-fatigued controls, where a significant
increase in the plasma pro-inflammatory cytokines IFN-c and
TNF-a were observed in the SCFS patients. Additionally, IL-4
was significantly increased in the SCFS group (Fig. 3).
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Autoimmun Highlights
A
0.25
SCFS
MCFS
Control
Concentration of
IL-4 (pg/ml)
*
0.00
SCFS
0.25
Control
SCFS
MCFS
Control
*
TNF- α (pg/ml)
Concentration of
B
MCFS
0.00
SCFS
C
MCFS
0.4
SCFS
MCFS
Control
IFN-γ (pg/ml)
*
Concentration of
Control
0.2
0.0
SCFS
MCFS
Control
Fig. 3 Plasma Cytokines in SCFS, MCFS and a non-fatigued control
group. The above bar graph is based on enzyme-linked immunosorbent assay (ELISA) assessments of seven plasma cytokines (IFN-c,
IL-1b/IL-1F2, IL-2, IL-4, IL-17, IL-6 and TNF-a). IL-4 (a), TNF-a
(b) and IFN-c (c) were significantly increased (P \ 0.05) in the SCFS
group in comparison to the MCFS and non-fatigued controls. Asterisk
denotes statistical significance where P \ 0.05. Results are represented as the mean ± SEM
Conclusion
This preliminary study is the first to examine and report
immunological disparities in severely affected CFS
patients characterised by significant decreases in NK lysis
and increases in KIR3DL1, IL-4, TNF-a and IFN-c.
The decreases in cytotoxic activity observed in the SCFS
and MCFS group were consistent with previous CFS NK
studies [34, 37, 69]. These NK disparities likely occur as a
consequence of paucities in lytic proteins including perforin
and granzymes and differential expression of their genes in
some CFS patients [37, 85]. These molecules are involved
in the granule-dependent cytotoxic pathway. Perforin is a
necessary component of this pathway as it facilitates the
entry of the granzymes into the target cell. In the target cell,
granzymes activate caspases, mitochondria-related apoptosis and reactive oxygen species, which induce apoptosis
[123]. Importantly, mice deficient in perforin experience a
substantial loss in cytotoxic activity. Reduced cytotoxic
activity permits the recurrence and prolonged survival of
various infections in the body possibly explaining the persistence of flu-like symptoms in the CFS patients. As correlations exist between cytotoxic activity and perforin in
CFS patients, a similar incidence may present itself in SCFS
patients perhaps at a more severe rate in comparison to the
moderately affected CFS population. Nonetheless, further
confirmatory studies are now required.
Significant increases in the expression of inhibitory
KIRs may correspond to the reduced NK cell lysis [124].
Specifically, the significant increase in KIR3DL1 may be
related to decreases in NK cell cytotoxicity of infectious
cells with Class I HLA expression [101]. KIR3DL1 is a
highly polymorphic inhibitory NK receptor and polymorphisms in its gene results in the generation of eight different KIR3DL1 allotypes that may be classified as having
high, intermediate or no surface expression with similar
affinity to bind antigens [102, 104, 105]. It associates with
antigens expressing HLA-B and having Bw4 specificity
[125]. Variations in the allotypes determine the response of
the KIR3DL1 to pathogens at the cell surface [104]. For
example expression of an inactivated KIRD3DL1 phenotype at the cell surface maybe subverted by ligands from
viral pathogens and this may be related to certain disease
presentations [126]. Similarly, polymorphisms within the
HLA-Bw4 may undermine recognition by KIR3DL1 [127].
Increases in KIR3DL1 have been associated with diseases
such as HIV and spondyloarthritis [66, 128]. KIR genes
have previously been investigated in CFS patients where
frequency of KIR3DS1 was significantly elevated in the
CFS patients in comparison to the non-CFS group [65].
Similarly, the incidence of KIR3DL1 and KIR3DS1 without HLA-ligand and HLA Ile80, respectively, was higher
among the CFS patients [65]. KIR3DL1 and KIR3DS1 are
encoded by the same gene [109], hence, these observations
implicate possible compromises to the genetic framework
that confers atypical properties on these receptors and their
allotypes inadvertently compromising cytotoxic activity.
123
Author's personal copy
Autoimmun Highlights
The increase in IFN-c did not correlate with an increase
in cytotoxic activity as cytotoxicity was reduced in the CFS
patients thus indicating that other cell types or cytokines
such as TNF-a may have contributed to the overall increase
in plasma IFN-c levels. The results on the cytokine studies
further highlight profound compromises in the immune
function of SCFS patients in comparison to the MCFS
patients. IFN-c and TNF-a activate macrophages and
CD8?T cells and provoke T helper 1-related immune
responses [129]. Persistent T cell and macrophage activation, decreased NK activity and impaired perforin function
is a hallmark of hemophagocytic lymphohistiocytosis
[130]. Hence, atypical immune activation may exist among
SCFS patients. Perhaps, cell-specific cytokine assessments
may provide superior in-depth analysis of cytokines in CFS
patients. Although, we have attempted to provide and
highlight cytokines in plasma this may still not be representative of the cytokine profile in CFS patients as the
source of most of these cytokines were not examined in this
study and thus remains unknown. Nonetheless, this is the
first study to report on cytokines in SCFS patients and may
serve as a platform for further studies.
Contrary to previous studies, the present study did not
demonstrate any significant reductions or changes in NK
phenotypes. CFS is a heterogeneous disease and different
subgroups of CFS patients may potentially express different distributions in immune cell phenotypes [55]. Incidentally, we have previously shown that alterations in NK
phenotypes are not consistent overtime but fluctuate and
are therefore poor indicators of immune function in CFS
patients [31]. Reduced NK lysis with concomitant increases in KIR3DL1 and cytotoxic-related cytokines is suggestive of impairments in the NK cell cytotoxic pathways,
in particular, the granule-dependent and -independent
pathways. Further studies are now required to elucidate the
mechanisms of these pathways in the CFS patients with
varying degrees of symptom severity. Importantly, KIR
receptors may be important biomarkers for the diagnosis of
CFS following thorough validatory studies to determine
their use in CFS diagnosis.
Conflict of interest E.W. Brenu, S.L. Hardcastle, G.M. Atkinson,
Mieke L. van Driel, Sanne Kreijkamp-Kaspers, K.J. Ashton, D.R.
Staines, S.M. Marshall-Gradisnik declare that they have no conflict of
interest.
Informed consent All procedures followed were in accordance
with the ethical standards of the responsible committee on human
experimentation (institutional and national) and with the Helsinki
Declaration of 1975, as revised in 2005. Informed consent was
obtained from all patients for being included in the study.
Animal studies
for this article.
123
No animal studies were carried out by the authors
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