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Etiology and Pathophysiology/Diagnostic

Peripheral blood leucocyte subclasses as potential


biomarkers of adipose tissue inammation and
obesity subphenotypes in humans
T. Pecht
1,2
, A. Gutman-Tirosh
1
, N. Bashan
1
and A. Rudich
1,2
1
Department of Clinical Biochemistry and
Pharmacology, Faculty of Health Sciences,
Ben-Gurion University of the Negev,
Beer-Sheva, Israel;
2
The National Institute of
Biotechnology (NIBN) in the Negev,
Ben-Gurion University of the Negev,
Beer-Sheva, Israel
Received 18 August 2013; revised 1 October
2013; accepted 18 October 2013
Address for correspondence: Dr A Rudich,
Department of Clinical Biochemistry and
NIBN, Faculty of Health Sciences, Ben-Gurion
University of the Negev, Beer-Sheva 84103,
Israel.
E-mail: rudich@bgu.ac.il
Summary
While obesity is clearly accepted as a major risk factor for cardio-metabolic
morbidity, it is also apparent that some obese patients largely escape this associa-
tion, forming a unique obese subphenotype(s). Current approaches to dene such
subphenotypes include clinical biomarkers that largely reect already manifested
comorbidities, such as markers of dyslipidaemia, hyperglycaemia and impaired
regulation of vascular tone, and anthropometric or imaging-based assessment of
adipose tissue distribution. Low-grade inammation, evident both systemically
and within adipose tissue (particularly intra-abdominal fat depots), seems to
characterize the more cardio-metabolically morbid forms of obesity. Indeed,
several systemic inammatory markers (C-reactive protein), adipokines (retinol-
binding protein 4, adiponectin) and cytokines have been shown to correlate in
humans with adipose tissue inammation and with obesity-associated health risks.
Circulating leucocytes constitute a diverse group of cells that form a major arm
of the immune system. They are both major sources of cytokines and likely also
of inltrating adipose tissue immune cells in obesity. In the present review, we
summarize currently available literature on classical blood white cell classes and
on more specic leucocyte subclasses present in the circulation in human obesity.
We critically raise the possibility that leucocytes may constitute clinically available
markers for the more morbidity-associated obesity subphenotype(s), and when
available, for intra-abdominal adipose tissue inammation.
Keywords: Cardio-metabolic risk, circulating leucocyte classes, metabolic syn-
drome, peripheral blood immune cells.
Introduction
Low-grade systemic inammation is now well-accepted to
accompany human obesity, and is thought to aetiologically
contribute to obesity-associated cardio-metabolic comorbi-
dities. The precise aetiology and relative contribution of
obesity-associated systemic inammation are not fully
characterized. It may involve both direct activation of
immune cells in the circulation, their recruitment into the
circulation, as well as the inammatory processes involving
immune cells within specic tissues, including the liver,
pancreas, muscle and adipose tissue. Of these, the adipose
tissue has been proposed both as an initiator and as a major
contributor to systemic inammation, and may therefore
serve to conceptualize the relations between specic organ
and systemic inammation in obesity. A detailed descrip-
tion of the evolution of the adipose tissue inammation
concept in obesity is beyond the scope of the present
review. Yet, it seems fair to say that adipose tissue inam-
mation obeys major paradigms of immune response (1),
such as the early involvement of neutrophils (2), the sub-
sequent recruitment of various lymphocyte populations (3)
obesity reviews doi: 10.1111/obr.12133
1 2013 The Authors
obesity reviews 2013 International Association for the Study of Obesity
and the ultimate recruitment and polarization of macro-
phages, as well as mast cells (4). Moreover, adipose
inammation in obesity may undergo dynamic transitions
from classical pro-inammatory cascades to inammation-
resolution and tissue remodelling processes (5,6).
The impact of systemic and adipose inammation in
human obesity, in particular intra-abdominal fat inamma-
tion, is exemplied by the highly intriguing subphenotype of
the insulin-sensitive (or metabolically healthy) obese (7).
This human obese subphenotype seems to escape the well-
documented associations between increasing body mass
index (BMI), systemic inammation, and cardio-metabolic
morbidity. Importantly, insulin-sensitive obese persons are
frequently characterized by a relative absence of intra-
abdominal fat inammation compared with BMI-matched
persons (7,8). However, because intra-abdominal fat is
not readily available for clinical assessment, and because
adipose-inltrating immune cells in obesity are largely
derived from bone marrows, a major gap of knowledge
is whether specic circulating leucocyte (sub)populations
could serve as markers of intra-abdominal fat inamma-
tion, systemic inammation, and thereby, of obesity-
associated cardio-metabolic risk.
Total leucocyte count has been recognized to correlate,
and even predict, cardiovascular disease already 40 years
ago. In addition, clinical markers of obesity, adiposity/fat
distribution (waist-to-thigh ratio), insulin resistance and
glycaemic control, have all been found to independently
contribute to the variance in white blood cell (WBC) counts
in the population (9,10). Yet, while inspiring research on
potential mechanisms in cardio-metabolic diseases, particu-
larly on the expanding role of inammation, this nding
has remained with limited use in clinical practice (11), for
several main reasons. First, WBC correlate with multiple
genetically, environmentally and even socially-determined
factors, including (among many others) sex, ethnic back-
ground, education level, height, smoking (active and
passive) and tness level. Second, the range of WBC counts
typically varies by no more than 20% among persons
stratied according to various obesity-related factors.
Although obesity was proposed as a cause of leucocytosis
(12), in most studies values are well within the normal
range, and with large variance, creating signicant overlap
between the comparison groups. This complicates the
ability to dene clinically meaningful cut-off values. Third,
despite a large body of data, even today it is still difcult to
assign WBC counts a true predictive value of future disease.
Most studies are cross-sectional, and in such association
studies WBC counts remain merely markers of a disease
state. Few longitudinal studies have demonstrated total
WBC counts at baseline as independent predictors for inci-
dent diabetes, deterioration of peripheral insulin sensitivity,
metabolic syndrome (MetS) or coronary artery disease
(1316). Yet, a more clinically signicant relative risk (or
hazard ratio) was mostly apparent in populations where
the absolute risk is rather low, such as in young, apparently
healthy, adults (13,14), as compared with more high-risk
groups (15,16).
One obvious direction to increase the clinical usefulness
of analysing circulating leucocytes to assess cardio-
metabolic risk in obesity is the assessment of specic leu-
cocyte classes. Information on the major leucocyte classes
(monocytes, lymphocytes, granulocytes) was greatly facili-
tated by automated differential blood counts technology.
However, even with those major classes of circulating leu-
cocytes, similar limitations to those described earlier for
total WBC counts still exist. Luckily, there is an explosive
increase in knowledge on the roles and pathways of inam-
mation and immunity in cardio-metabolic diseases, as well
as on subclasses of circulating blood leucocytes within each
of its major groups. These are dened by a growing number
(although frequently still debated) specic and semi-specic
surface markers that can be used to recognize, quantitate
and isolate such cell subpopulations by robust technol-
ogies, as uorescence-assisted cell sorting. Collectively, it
seems that we are currently at a stage of a growing effort
to determine the diagnostic, prognostic and potentially
mechanistic role(s) of highly specied subpopulations of
circulating blood leucocytes in cardio-metabolic diseases.
Hopefully, results of such efforts will complement ndings
over recent years, in which cytokines levels have dominated
the assessment of inammatory tone and its potential con-
tribution to disease.
Literature search approach
Studies included in this review were those addressing
the potential of different leucocytes populations/sub-
populations, as dened in Table 1 and Fig. 1, to associate
with human obesity in clinical cross-sectional and longitu-
dinal studies (Tables 2 and 3). More specically, in cross-
sectional studies, for each leucocyte type we considered the
following levels of association: (i) Is it different among
obese versus non-obese persons? (ii) Is it associated
with obesity-related phenotypes, including bio-markers
of obesity-associated morbidity, the composite denition of
Table 1 The major classes and subclasses of circulating leucocytes
discussed in this Review
Circulating
cell type
Normal count
(count per mm
3
) (103)
Monocytes 300600
Neutrophils 3,0006,000
Eosinophils 150300
Basophils 0100
Lymphocytes 1,5004,000
2 Circulating leucocytes and obesity subphenotypes T. Pecht et al. obesity reviews
2013 The Authors
obesity reviews 2013 International Association for the Study of Obesity
the MetS, and healthy/insulin sensitive obese versus insulin-
resistant obese. (iii) Is a specic leucocyte class associated
specically with adipose tissue inammation? For longitu-
dinal studies, we assessed if baseline leucocyte class counts
were predictive of subsequent obesity-related outcome(s).
Alternatively we assessed whether counts dynamically
changed in response to weight loss intervention in parallel
to improvement in clinical parameters of obesity-associated
comorbidity.
Circulating monocytes
Total circulating monocytes
Increased interest in circulating monocytes has emerged
since the discovery of enhanced adipose tissue macrophage
(ATM) inltration in obesity in mice (17,18) and later in
humans (19,20), and the suggestion that these cells were
likely of bone-marrow origin (i.e. monocytes from the
circulation that differentiate in adipose tissue into macro-
phages). Higher mean monocyte counts, typically by
1318%, were reported in overweight and obese compared
with lean persons (21), to correlate with BMI (22) and with
higher prevalence of the MetS (16). Yet, others did not nd
such correlation (23,24), and even reports of negative asso-
ciation between the percentage of total monocytes and BMI
could be found (25). Nevertheless, potential confounders
were also identied in some studies: monocyte counts
correlated with BMI only in women, but not in men (22),
in whom, in another study, adjusting for tness level
abolished the crude correlation between monocytes and
BMI (26).
Monocytes gene expression and expression of specic
surface molecules were demonstrated to differ between
obese and non-obese persons, and to correlate with obesity-
related clinical abnormalities: a higher (approximately
twofold) activated state (integrin CD11b surface expres-
sion) was noted in obese compared with non-obese, and the
level of CD11b expression correlated with insulin resist-
ance (higher homeostatic model assessment insulin resist-
ance [HOMA-IR] ) (25). Following the notion that ATMs
in obesity may undergo a phenotypic switch from alterna-
tively activated resident macrophages (M2) to classically
activated, pro-inammatory macrophages (M1) (27),
M1\M2 terminology has also been adopted to characterize
Figure 1 Relative distribution of circulating leucocytes subclasses.
(a) The distribution of the major circulating leucocyte classes (103). (b) the distribution of monocyte subclasses by surface expression of CD14 and
CD16 (104). (c) the distribution of the lymphocyte classes (105) and CD4+ T cells subclasses (106,107).
obesity reviews Circulating leucocytes and obesity subphenotypes T. Pecht et al. 3
2013 The Authors
obesity reviews 2013 International Association for the Study of Obesity
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4 Circulating leucocytes and obesity subphenotypes T. Pecht et al. obesity reviews
2013 The Authors
obesity reviews 2013 International Association for the Study of Obesity
circulating monocytes. The underlying notion was
perhaps that ATM subpopulations are derived from pre-
determined specic circulating monocytes. Higher expres-
sion of the M1 markers tumour necrosis factor (TNF)
and interleukin (IL-6), and lower expression of the M2
marker IL-10 were found in monocytes from obese non-
diabetic persons compared with lean controls (28). Addi-
tionally, comparing diabetic-obese to non-diabetic obese
persons, lower mRNA expression of the M2 markers
CD163 and of IL-10 were found in the former group,
suggesting that lower M2 markers correlate with obesity-
associated morbidity (28). Further following this notion, in
a multivariate regression analysis, the mRNA expression of
the M1-related markers TNF and IL-6 was associated with
BMI, while IL-6 also associated with low-density lipopro-
tein cholesterol levels. The two M2-related markers (CD163
and IL-10) also correlated with clinical parameters: lower
IL-10 mRNA expression associated with higher diastolic
blood pressure, glycated haemoglobin (HbA1C) and
triglycerides (TG), and lower CD163 expression with higher
fasting insulin concentrations, HbA1C and pulse wave
velocity (a measure of arterial stiffness). In vitro, monocytes
exhibited decreased responsiveness to induced differentia-
tion into an M2 phenotype (lower M2 gene expression) in
both diabetic and non-diabetic obese compared with lean
women (29). This latter nding suggests that ATM polari-
zation might be already programmed in the circulating
monocytic precursor cell, a notion also supported by func-
tional in vitro assays: Increased reactive oxygen species
(ROS) and activation of endoplasmic reticulum stress were
demonstrated in circulating monocytes from obese non-
diabetic compared with lean persons (30); peripheral
blood mononuclear cell (PBMC)-derived monocytes from
obese persons with no comorbidities exhibited signi-
cantly increased basal and lipopolysaccharide (LPS)-
induced TNF production compared with non-obese,
correlating with metabolic parameters, TGlevels and serum
insulin concentration (31).
Interestingly, assays addressing monocytes functions
more related to endocrine-metabolic dysfunction in obesity
have also demonstrated links with obesity: intracellular
processing of the insulin-receptor by monocytes revealed
that receptor recovery, release of internalized insulin
(insulin retro-endocytosis) and its intracellular degradation
were decreased in both obese diabetic and non-diabetic
patients compared with healthy persons (32). Impaired
only in the diabetic-obese compared with healthy lean con-
trols was monocytes insulin-induced receptor internaliza-
tion (32,33). Combined with altered monocytes functions
more classically related to their immune functions, these
perturbations in insulin-insulin receptor dynamics may
suggest a possible link between endocrine and immune
dysfunction in obesity that can be detected and studied at
the level of circulating monocytes.
The mechanisms for the possible circulating monocytes
programming in obesity and obesity-associated diabetes is
a growing area of interest: micro-RNAs have been impli-
cated in the deregulation (elevated expression) of some M1
markers in circulating monocytes in obesity: miR-181a,
181b and 181d were identied as possible regulators
of the toll-like receptor (TLR)/nuclear factor kappa-light-
chain-enhancer of activated B cells (NFB) signalling
potentially by targeting TLR4, the NFB adapter molecules
TRAM, TAK1 and TAB2, and the inammatory cytokine
TNF. The expression level of these miRs in CD14
+
PBMC (considered as monocytes) negatively correlated
with systemic inammation (circulating IL-6 and high-
sensitivity C-reactive protein [hsCRP] levels) in a cohort
comparing morbidly obese to lean persons (34). In the same
study, another cohort comprised of high-risk patients for
coronary artery disease who underwent coronary angiogra-
phy exhibited lower levels of miR-181a, but not of miR-
181b and miR-181d. This de-regulated expression of miR-
181a associated with a higher number of MetS components
and with angiograph-proven coronary disease, even after
adjustment for traditional risk factors (34). Along the
same line, another miRNA that was identied to decrease
in monocytes from obese compared with lean persons
was miR-146b-5p, which suppresses the IL-1 receptor-
associated kinase (IRAK)/NFB pathway adapter molecules
IRAK1 and TNF receptor-associated factor-6 (TRAF6) (35).
Specic circulating monocytes subpopulations
Beyond total circulating monocytes, it has become
apparent in recent years that human blood monocytes
constitute a heterogeneous cell population that can be
subdivided according to the surface expression pattern of
CD14 the LPS co-receptor (with TLR4 and MD-2), and
CD16 the low-afnity FcIII receptor. Three human
monocyte subpopulations have been recently dened
based on these surface markers: CD14
+
CD16

(classical
monocytes), CD14
+
CD16
+
(intermediate monocytes),
and CD14
dim
CD16
+
(non-classical monocytes) (Table 1,
Fig. 1). The latter two groups are sometimes analysed
together (i.e. CD16
+
monocytes).
CD16
+
monocyte counts were reported to be higher in
obese compared with lean persons (23,36,37), and to cor-
relate with BMI (23). Moreover, intima-media thickness
(IMT) of the carotid artery, a measure of subclinical
atherosclerosis, correlated with higher CD16
+
monocyte
counts, an association that was lost when adjusting
for BMI, or the Framingham cardiovascular risk score.
Complementarily, BMI correlation with IMT was attenu-
ated when adjusting for the CD16
+
monocytes count, sug-
gesting that the increase in CD16
+
monocyte counts is in
the same causal pathway linking obesity to subclinical
atherosclerosis (23). Importantly, circulating CD16
+
mono-
cytes correlated with subcutaneous ATMs (CD68
+
cells),
obesity reviews Circulating leucocytes and obesity subphenotypes T. Pecht et al. 5
2013 The Authors
obesity reviews 2013 International Association for the Study of Obesity
both of which were elevated in a cohort of normoglycaemic
obese versus lean controls (37), indicating that specic
monocyte subclass(es) may constitute a marker for adipose
tissue inammation, and thus potentially, for the tendency
to develop a more cardio-metabolic morbid form of
obesity.
When considered separately, CD14
+
CD16
+
and
CD14
dim
CD16
+
were both increased, by 70100%, in obese
compared with lean persons, and showed an association
with glucose tolerance, insulin sensitivity (glycaemia,
insulin, HOMA-IR), inammation markers (hsCRP) and
adiposity (BMI and fat mass) (38). Yet, CD14
+
CD16
+
(intermediate) monocyte subpopulation did not correlate
with BMI (23), whereas CD14
dim
CD16
+
(alternative)
monocytes were higher in obese diabetic compared with
non-diabetic (38), and their counts correlated with param-
eters indicating higher risk for cardio-vascular disease (age,
blood pressure, total cholesterol and [lower] high-density
lipoprotein [HDL] ) (23).
As for CD14
+
CD16

(classical) monocyte subpopulation,


available reports are inconsistent, reporting either
decreased or no change in obese compared with lean
persons (23,38), possibly mirroring the reported inconsist-
ent associations between obesity and total monocyte counts
which were mentioned above (classical monocytes are the
most abundant monocyte subpopulation). Yet, in children,
both CD14
+
CD16

(39,40) and CD14


+
CD16
+
(40) mono-
cyte subpopulations, as well as their surface CD11b
expression all correlate with BMI (40), suggesting that
childhood obesity may manifest a stronger link between
circulating monocyte subpopulations and obesity.
Further support by follow-up studies
Moving from cross-sectional to dynamic/follow-up studies,
monocytes seem to respond, at several levels, to weight-loss
interventions: Weight loss induced during 6 weeks of very-
low-energy diet (VLED) intervention was accompanied by
a decrease in monocyte counts in normoglycaemic obese
persons compared with baseline (41). Moreover, micro-
RNA (miR-)181a, 181b, and 181d expression, which
was found to be decreased in monocytes of morbidly obese
patients compared with lean persons, normalized 3 months
after surgery-induced weight loss (34). Along the same
line, in vitro LPS-induced TNF production by PBMC-
derived monocytes was lower in obese persons with
no comorbidities after VLED compared with baseline
(although remained elevated compared with non-obese
persons) (31).
Similar ndings were reported with specic monocytes
subpopulations: the percent of CD16
+
monocytes out of
total monocytes decreased in morbidly obese persons
twelve months after Roux-en-Y gastric bypass (RYGB)
surgery (36). The extent of decrease correlated with the
degree of changes in weight, fat mass (BMI) and TG (38).
Interestingly, a decrease specically in the CD14
dim
CD16
+
percentage was dependent on at least 5% weight loss,
whereas the change in the CD14
+
CD16
+
subpopulation did
not correlate with changes in fat mass (38). In addition, a
decrease in carotid IMT three months after surgery-induced
weight loss was reported to associate with a decrease in
CD14
+
CD16
+
monocytes, an association attenuated by
controlling for the change in BMI or fat mass (38).
Collectively, while several studies suggest a mild
increase in total circulating monocyte counts in obesity,
this has not been a universal nding in some studies
involving smaller number of participants (Table 2). Yet,
several specic markers of monocyte activation state
exhibit a more pronounced change in obese compared
with non-obese/healthy controls, and importantly, such
monocyte programming may correlate with systemic
insulin resistance, and be normalized by weight-loss inter-
vention. An additional possible means of increasing the
potential signicance of monocyte measurements is by
assessing specic subpopulations, that may not only more
strongly associate with obesity and related cardiovascular
morbidity, but may also causally mediate the link between
obesity and atherosclerosis, possibly by signifying the
degree of AT inammation.
Circulating neutrophils
In comparison with the interest in circulating monocytes
that emanated from the discovery of ATMs in obesity,
neutrophils have only recently been recognized to inltrate
the adipose tissue of obese mice, and potentially contribute
to obesity-induced dysmetabolism (2,42,43). In humans,
obesity has been proposed as a cause of persistent
neutrophilia, manifesting by elevated neutrophils (and
total WBC) counts (12). Yet, even within the normal range,
neutrophil counts correlate with BMI in most studies
(11,44), are increased in patients with the MetS compared
with control non-MetS persons (11,44,45), and correlate
with insulin resistance markers (11,44) and systemic
inammation (hsCRP) (44). They were even proposed as
the leucocyte population that predominates the elevated
total WBC observed in MetS compared with non-MetS
patients even when adjusting for age, sex, BMI and waist
circumference, statistical models in which association with
monocytes was lost (44). Children exhibit a similar asso-
ciation of BMI and adiposity with increased neutrophil
counts, with the highest correlation coefcients compared
with any other leucocyte population assessed (46). In
Byukkaya et al. (45), 70 MetS (not necessarily obese)
persons exhibited signicantly elevated (by somewhat
unusually 25% higher) neutrophil counts compared with
age- and sex-matched non-MetS controls, along with a
decrease in lymphocyte counts (which is not a consistent
nding see section dealing with lymphocytes). Putting
6 Circulating leucocytes and obesity subphenotypes T. Pecht et al. obesity reviews
2013 The Authors
obesity reviews 2013 International Association for the Study of Obesity
these somewhat unique characteristics of this cohort aside,
a neutrophil-to-lymphocyte (NLR) ratio of >1.84 (sug-
gested to represent a high pro-inammatory load) had a
sensitivity (true-positive rate) of 92.8% and a specicity
(true-negative rate) of 61.9% for MetS. NLR > 1.84 was
independently predicted by hsCRP and fasting glucose
levels in a multivariate model that also included HDL,
waist circumference, BMI and TGs (45).
Beyond cross-sectional associations, follow-up studies
also demonstrate dynamics in neutrophil counts related to
obesity and/or its outcomes. In the recent large-scale
PREDIMED study higher baseline neutrophil levels or an
increase in neutrophil counts during >3.5 years mean
follow-up were independently associated with 30%
elevated risk of incident MetS among cardiovascular
disease-free persons 55 years of age or older (16). Although
these associations were also seen with total WBC and
with some of the other leucocyte populations, neutrophils
seemed as the strongest and most consistent leucocyte class,
particularly predicting the appearance of dyslipidaemic
components of the MetS during follow-up (16). Both life-
style (diet exercise) (47) and surgical interventions (11)
were reported to decrease neutrophil counts. Agreater drop
in BMI may be the strongest independent predictor of a
decline in neutrophil (or polymorphonuclear) counts, even
when adjusting for markers of insulin resistance and/or
when comparing MetS with non-MetS patients (11). Inter-
estingly, in post-menopausal women randomized for 1 year
of either hypocaloric diet alone, exercise, or their combina-
tion, diet (and not exercise) was the driver for a decline in
neutrophil counts (and other inammatory markers) (47). It
is worth mentioning, though, that overall the mean change
in neutrophil counts was typically 10% of baseline.
Neutrophils functions have been largely reported to be
altered in obesity and to respond to interventions, but the
emerging picture is complex, suggesting obesity-associated
activation and/or dysfunction of these cells (Table 2). This
may reect, beyond generic issues such as the specic
patients characteristics, factors more specic to neutro-
phils biology and to howit is studied: neutrophil activation
is a multifaceted process that involves multiple stages
(priming versus activation) and specic changes in cellular
functions (exposure and activation of adhesion molecules,
production and secretion of secreted products as diverse as
lipid mediators, cytokines, enzymes and ROS to name just
a few). On top of this complexity are different methodol-
ogies used to estimate specic neutrophils functions. Given
this complexity, we provide next few examples of studies
reaching the conclusion of either decreased or activated
neutrophils activity. For the former, neutrophils from
severely obese patients were shown to express lower surface
levels of CD62L, an L-selectin that mediates the initial
tethering and rolling of leucocytes on the endothelial
surface (36), a defect corrected 6 months following
surgery-induced weight loss. Contrasting the viewof dimin-
ished neutrophil function in obesity, surface expression of
neutrophil CD66b, considered a marker of neutrophil acti-
vation (48), was found to be elevated in a small number of
severely obese versus controls (49). This corresponded to
increased circulating levels of the neutrophil secreted prod-
ucts myeloperoxidase and calprotectin, the second of which
signicantly decreased in the obese patients 2 years after
surgery-induced weight loss (49). Consistently, circulating
neutrophil elastase levels were increased in correlation with
BMI in a recent publication (50), although this was not
found to associate with obesity in an earlier study (51). LPS
or formylmethionylleucylphenylalanine-induced IL-8
(CXCL8) production by neutrophils were increased in over-
weight and in obese patients compared with normal BMI
controls (52).
A potential reconciliation for the mixed conclusions
about the activation state of neutrophils may be offered by
a comprehensive analysis of several neutrophil functions
(albeit in a rather small group) of morbidly obese patients
(53). Neutrophils from obese patients had mildly dimin-
ished bacterial ingestion capacity, and stimulus-activated
ROS production, with no apparent effect of obesity on
chemotaxis. Impaired phorbol12-myristate-13-acetate-
induced ROS production was more severely observed in
morbid obese patients in the higher range of BMI (i.e.
>50 kg m
2
). Yet, concomitant to this dysfunction, basal
(non-stimulated) ROS production was signicantly elevated
in morbidly obese compared with controls (53). More-
over, neutrophils from morbidly obese patients exhibited
increased sensitivity to LPS-induced inhibition of neutrophil
apoptosis, potentially explaining prolonged neutrophil
half-life, particularly in light of the proposed low-level
endotoxaemia proposed to occur in obesity (54,55). Col-
lectively, it would seem that obesity associates with a
complex alteration of neutrophils inammatory function,
potentially with partial, basal, pro-inammatory activation
and possibly diminished ability to fully activate pro-
inammatory functions in response to stimuli. Although the
latter may theoretically provide mechanistic basis for
increased risk of infection in obesity, some investigators
reporting impaired neutrophil functions in even morbid
obesity viewed them as unlikely to account for grossly
impaired immune function (53).
There are multiple factors that have been proposed to
mediate the aforementioned altered neutrophil functions
in obesity, but most notable in human studies and speci-
cally for neutrophils are adiponectin and lactoferrin.
The obesity-associated decrease in the adipocyte-derived
hormone adiponectin was proposed to sensitize neutro-
phils to stimulus-induced CXCL8 secretion in obesity
(52). Interestingly, lactoferrin, another protein with anti-
inammatory effects, was found to be mildly decreased in
persons with dysglycaemia/diabetes, both at the circulating
obesity reviews Circulating leucocytes and obesity subphenotypes T. Pecht et al. 7
2013 The Authors
obesity reviews 2013 International Association for the Study of Obesity
level and ex vivo in LPS-stimulated whole blood (56). Both
factors may link obesity with neutrophils pro- inamma-
tory and/or hyper-activatable state.
Other granulocytes: circulating basophils
and eosinophils
Obesity is increasingly recognized to associate with allergy
or asthma (57,58), providing a natural potential associa-
tion with basophils and eosinophils that are inherent
components of allergic inammation. Both of these popu-
lations constitute a small fraction of total circulating WBC
(Table 1, Fig. 1) and a minority among the granulocytes
(neutrophils being the vast majority of that group). Overall,
current information on circulating blood basophils and
eosinophils in human obesity-associated inammation is
limited and rather inconsistent.
Basophils play an important role in the initiation and
propagation of immediate-type hypersensitivity reactions
by binding antigen-specic immunoglobulin E (IgE) that
triggers their activation, granule exocytosis, and the release
of histamine and other allergic-reaction mediators. Cur-
rently there are no reports on increased presence of
basophils in AT. Nevertheless, leptin receptor on human
basophils was found to mediate enhanced survival, migra-
tion, activation (CD63 levels), degranulation and cytokine
synthesis (59), suggesting a role for obesity-associated
hyper-leptinaemia in circulating basophils activation
state. In the circulation, basophil counts were reported to
either associate with BMI/MetS (26,60,61), not associate
(16,24,62,63) or to even negatively associate with it
(64): positive associations in both men and women were
observed between basophil counts and the presence of the
MetS after adjusting for age, smoking, alcohol intake, edu-
cation level and household income. Yet, the association was
lost when other leucocyte counts (total WBC, neutrophils,
lymphocytes, eosinophils and monocytes) were included
in the model (61). In two studies, an increased absolute
basophil count (26,60) and basophils percentage of total
WBC (60) were reported to correlate with higher BMI. In
contrast, others reported no correlation between basophil
counts and BMI, and no difference between the obesity
groups among female students (62). Additionally, in cross-
sectional studies on healthy middle-aged men (24,63) and
women (63), diabetic persons (16,24) and persons with
cardiovascular risk (16), higher basophil counts were not
associated with either MetS prevalence (16,63) (while
all other leucocytes classes did) (16), or the number of
the MetS criteria (24,63). Finally, a negative correlation
between basophils counts and BMI, waist circumference
and total adiposity (calculated using computed tomogra-
phy) was reported in adolescent women after adjusting for
age, blood pressure, total cholesterol, HDL-cholesterol, TG
and fasting glucose (64).
Beyond peripheral basophil counts, serum concen-
tration of IgE was used to estimate basophil activation. IgE
is the immunoglobulin subtype with the lowest abundance
in plasma, and is tightly regulated in healthy persons.
Increased levels of total IgE (65,66) or specic IgE (66,67)
were demonstrated in obese, as compared with normal
weight children (65) and adults (66,67). Yet, others
reported no association between total or specic IgE and
BMI in young adults (68).
Eosinophils have been proposed to regulate adipose
tissue inammation by maintaining ATMs in an alter-
native activation (M2) state via IL-4 production (69). Yet,
as with basophils, there are conicting reports on whether
circulating eosinophil counts associate with human obesity:
several studies suggest an absence of association between
eosinophil absolute counts or percentage and BMI/MetS
(21,24,26,60,63). In contrast, in a small trial among
morbidly obese (BMI > 40) persons with a variety of
comorbidities, eosinophil percentage of total leucocytes
were increased approximately fourfold compared with the
lean control group with no comorbidities (36). Yet, this
elevated eosinophil percentage remained unresponsive
during 12-month follow-up after RYGB surgery, when
other parameters (like CD62L surface expression on
neutrophils and monocytes) were normalized (36). Also
paralleling ndings in basophils, leptin was shown to
enhance eosinophils functions and survival, offering a
putative mechanism linking obesity with exacerbation of
asthma (70).
Circulating lymphocytes
Lymphocytes are a class of immune cells which is further
divided into three major subclasses: T cell, B cells and
natural killer (NK) cells. Despite their heterogeneity, total
circulating lymphocytes are easily measured in routine dif-
ferential WBC counts, and can be readily isolated from
whole blood. Hence, we rst briey review lymphocytes
relevance in obesity as a single group, followed by consid-
eration of specic lymphocyte subpopulations.
Total circulating lymphocyte counts
Higher lymphocyte counts were reported in cross-sectional
studies to correlate with elevated BMI (11,16,60,61,71),
even after adjustment for age, sex (60,71), smoking (71) or
ethnicity (60). Moreover, total lymphocyte counts associ-
ated with higher MetS prevalence (16,61), increased
number of positive MetS criteria (24,61) and correlated
with the various individual MetS parameters or with higher
fasting insulin or HOMA-IR (11,24). Beyond cross-
sectional analyses, in the large longitudinal PREDIMED
study, a multivariate logistic regression analysis (although
not Cox regression) revealed that persons classied in
the top quartile of lymphocyte counts at baseline (i.e.
8 Circulating leucocytes and obesity subphenotypes T. Pecht et al. obesity reviews
2013 The Authors
obesity reviews 2013 International Association for the Study of Obesity
2.55 10
9
cells per L versus 1.54 10
9
in the lower
quartile) had a higher risk of incident MetS during a mean
follow-up of 3.9 years, in particular, development of the
hyper-TG and high-fasting glucose criteria (16). Addition-
ally, surgery-induced weight loss associated with a decrease
in lymphocyte counts in obese persons (11,72). In particular,
greater reduction in total circulating lymphocyte counts
were apparent in those with higher-fasting insulin at base-
line, and those with a greater percentage of weight reduction
(11). Despite the highly suggestive studies mentioned earlier
for a positive association between total circulating lympho-
cytes and obesity or markers of comorbidity, this has not
been reported by all investigators or with all markers
(11,21,22,46) (Table 2). Functionally, LPS-stimulated pro-
duction of inammatory cytokines by lymphocytes was
similar in overweight and lean young adults (73).
T cells
T lymphocytes, commonly identied by expressing CD3,
have been recently implicated in obesity-related AT inam-
mation (1). They were reported to inltrate AT prior to
macrophages, and to contribute to the development of
insulin resistance (74). In humans, AT T lymphocytes were
found to increase in obese compared with lean persons
and to correlate with BMI (75). However, circulating T cell
measures have yielded conicting reports: Total T cell
counts were found to increase by 1550% in morbidly
obese compared with lean and to correlate with adiposity
and/or fat distribution measurements in both adults,
women-only cohort and in children (46,62,76,77). Several
studies addressing the dysmetabolism that accompanies
obesity suggest that CD3+ Tcell counts may be linked to the
metabolic state. Higher CD3+ T cell counts were demon-
strated to associate with MetS and with specic MetS risk
factors (higher TG, lower HDL) (78) and to correlate with
the number of MetS risk factors in men (24,78). On the
other hand, CD3+ T cell counts were increased only in
obese, but not in morbidly obese, compared with lean
persons (22), were similar among obese and non-obese
children (79) and adults (25), and did not correlate with
BMI in female students (62). Furthermore, CD3+ T cells
percentage was the same between obese, lean and formerly
obese women after gastric banding surgery (80), and was
even decreased (by 20%) in obese compared with lean
persons in one study (31). In the small longitudinal arm of
this last study, weight loss induced among obese patients by
very-low-calorie diet (VLCD) was associated with increase
in CD3+ T cells counts (31). This energy-restriction effect
on elevation of T lymphocytes was also noted in their
measure of percentage from total WBC in obese patients
with T2D or impaired glucose tolerance (81). Alongside
studies assessing CD3+ T cell counts and/or percentage,
studies evaluating T cell functions and activation state have
also yielded variable results. Obese persons exhibited lower
blastogenic activity compared with lean controls, a function
which was increased following a VLCD induced-weight
loss (31). In contrast, T cell activation state, as indicated
by surface expression of CD25 (but not of CD69), was
reported to be increased in obese, although not in metaboli-
cally healthy overweight, compared with lean persons (25).
Yet, expression of both CD25 and CD69 decreased follow-
ing VLCD + surgery-induced weight loss, with CD25 drop-
ping by more than threefold, comparable with lean controls
(81). Using a different T cell activation marker, HLA-DR
(MHCclass II cell surface receptor), suggested no difference
between obese and lean persons, and no correlation with
BMI or change induced by VLCD-induced weight loss
(80).These overall conicting data about associations
between circulating total Tcell counts and obesity and/or its
comorbidities may reect, beyond differences in patient
cohorts and approaches to estimate T cell functions, also
the heterogeneity of the T cell population. Historically, T
cells were subdivided into CD8+ or CD4+ expressing cells.
This initial subdivision was soon realized to poorly repre-
sent the diversity of T cell subpopulations. Here, we will
rst review current information on obesity and circulating
cytotoxic T cells (CTL), cells characterized by the expres-
sion of CD8, a co-receptor of the T-cell receptor, which
mediates efcient cellcell interactions; we will then discuss
jointly the CD4+ T cell subpopulations, followed by sepa-
rate discussion of helper T cells (Th cells) and regulatory T
cells (Tregs). Detailed description of the unique functions of
these populations in immune responses is beyond the scope
of this review. Yet, it is worth mentioning that the Th
subpopulation designated Th1 and CTLs are mostly con-
sidered to promote pro-inammatory response, Th2 and
Treg are thought to suppress or limit it. Subsequent sections
further discuss how these specic T cell lymphocyte
subpopulations in circulating blood associate with obesity,
comorbidity and AT inammation.
T cells subpopulations: circulating CD8+ CTL (Table 3, i).
Cytotoxic CD8+ T cells were suggested to inltrate adipose
tissue in both mice and humans, and to promote the
(subsequent) recruitment, differentiation and activation of
macrophages (3). Inthe circulationinhumans, either absolute
and/or percent of CD8+ T cell counts were reported to be
reduced with obesity (80) (and Table 3) although a larger
number of publications suggest decreased percentage of
CD8+ T cells from total lymphocytes (73,82,83). Yet, others
reported either no difference (77) or increased CD8+ T cell
counts in morbid obesity (84) (Table 3). This may be related
to the elevated in vitro proliferative capacity of CD8+ T cells
isolated from morbidly obese compared with lean persons
(76). Association between CD8+ T cells and obesity subphe-
notypes were also proposed: CTL were less abundant among
obese-unhealthy compared with healthy obese persons (83).
In a large study including men, only higher CD8+ T cells
obesity reviews Circulating leucocytes and obesity subphenotypes T. Pecht et al. 9
2013 The Authors
obesity reviews 2013 International Association for the Study of Obesity
counts associated with the presence of the MetS, correlating
with the number of positive criteria for the MetS, specically
the hypertension and dyslipidaemic components (78).
Despite these reported alterations in CD8+ T cell
subpopulation with obesity, following weight loss no
change was reported in their percentage (81) or counts (31)
(induced by surgery or VLCD, respectively). Collectively,
although the majority of studies appear to indicate lower
absolute or relative circulating CD8+ T cells in obesity,
the lack of evidence for a change in these parameters
with weight-loss intervention questions the likelihood of
their signicant causal role in obesity-associated cardio-
metabolic morbidity, or at least in improvements in risk
factors following weight-loss interventions.
T cells subpopulations: circulating CD4+ (Table 3, ii). Cir-
culating T CD4+ cell counts were reported to be elevated in
morbidly obese (60,76,82,84), obese (22,62,80,84), and in
overweight (84) compared with lean persons, and to cor-
relate with BMI (76,82). CD4+ T cells also showed corre-
lations with waist-to-hip ratio (WHR) in female students
(62) and with adiposity (percent body fat) in children (46).
Relating to obesity-associated cardio-metabolic morbidity,
CD4+ T cell counts were reported to correlate with insulin
sensitivity (fasting insulin levels and glucose-to-insulin
ratio) in non-diabetic morbid obese women (76), and in
men with increased prevalence of MetS, specic MetS
parameters (hypertension, high TG, low HDL) and with
the number of positive MetS criteria (78). The observed
increase in CD4+ T cell numbers might be explained by an
increased capacity for proliferation as demonstrated in
vitro (76). This altered function of circulating CD4+Tcells
could not be attributed to specic dominant antigen(s), as
the T cell receptor repertoire was diverse. In contrast,
CD4+ T cell blastogenic capacity was lower in obese
compared with non-obese persons, and was increased after
VLCD-induced weight loss (31).
It is worth noting that unlike the fairly consistent cross-
sectional associations between circulating CD4+ cell counts
and obesity, the relative abundance (percentage) of this cell
type shows weaker associations with obesity: CD4+ T cell
percentage of total lymphocytes was similar between obese/
morbidly obese and lean persons (22,73,83), and no cor-
relation was found with BMI among adults (22,62). Similar
results were obtained in children, in whom no difference
was found in circulating T CD4+ cells percentage (or
counts) between obese children with MetS and lean
children (85,86).
Finally, circulating CD4+ T cells may not be responsive
to weight-loss intervention: CD4+ T cell counts were unal-
tered between obese and formerly obese women who (his-
torically) underwent bariatric surgery and nutritional
intervention (80). Even more directly, CD4+ T cell percent-
age of lymphocytes did not change after surgery-induced
weight loss (81).
T cells subpopulations: circulating Th1 and Th2 (Table 3,
iii). CD4+ Th cells are considered the coordinators of the
immune response, with two main subsets Th1 and Th2
lineages (4). Th1 cells engage in a pro-inammatory immune
response, involving secretion of cytokines such as
interferon- (IFN) and participation in cell-mediated
immune responses, while Th2 cells are largely anti-
inammatory cells, producing IL-4 and IL-13 and promot-
ing the humoural immune response (4). In AT, the main
paradigm states that Th1 cells increase with obesity, while
Th2 (and Tregs, see next section) decrease, shifting the
delicate balance between pro- and anti-inammatory
immune cells within the tissue towards a more pro-
inammatory tone (3). To our knowledge, in the circulation,
Table 3 Summary of studies assessing circulating T cell subclasses in human obesity
Level of association with
obesity
i. ii. iii. iv.
Cytotoxic T cells (CD8+) CD4+ Th1,Th2,Th1/Th2 Tregs
i. Association of leucocyte
number or % with body mass
index/adiposity (CS)
(+) (84): (+) (22,46,60,62,76,80,82,84): (+) (25,76,79): (+) (76):
(0) (77): (0) (22,62,73,83,85,86): (0) (76): (0) (86,90):
() (54,73,79,80,82,83): () (31): () (90): (%of CD4+)
Association with functional or
activation markers
(+) (76): (+) (76):
() (85):
ii. Association with obesity
subphenotypes, like MetS
and insulin resistance (CS)
(+) (73): (+) (76,78): (+) (25,76,79): (85) (90)
() (83):
iii. Follow up/intervention
longitudinal studies
(+) (81):
(0) (31,81): (0) (80,81):
() (31):
iv. Correlate with adipose
tissue inammation
10 Circulating leucocytes and obesity subphenotypes T. Pecht et al. obesity reviews
2013 The Authors
obesity reviews 2013 International Association for the Study of Obesity
Th1 and Th2 absolute counts have not been frequently
reported, although more information is available on their
relative abundance (percentage of CD4+ Tcells). Th1 counts
(identied as CD4
+
IFN
+
PBMCcells) did not differ between
morbidly obese and lean persons (76), although others
reported a correlation with BMI and measures of central
adiposity (waist and WHR) (25). On the other hand, the
percentage of Th1 cells (identied as CD4+ IFN secreting
PBMC) was found to increase (79) in obese compared with
lean, and to correlate with insulin resistance (HOMA-IR
(25,79) and fasting insulin (79) ) and leptin (79), in both
children (79) and adults (25). Even more provoking, Th1
cells percentage correlated with the presence of non-
alcoholic steatophepatitis only among obese children, but
did not correlate with adiposity (standard deviation-BMI
and total body fat mass) nor with systolic blood pressure
(79). Importantly, Th1 cells percentage decreased in
response to VLCD-only or VLCD + surgery-induced weight
loss in morbidly obese men (81).
Circulating Th2 cell counts (identied as CD4
+
IL-4
+
PBMC cells) were reported to increase in morbidly obese
persons and to correlate with BMI (76), although no dif-
ference was found in the percentage of Th2 cells between
obese, metabolically healthy overweigh and lean persons
(25). Th1\Th2 ratio, used as an index of pro- versus
anti-inammatory T cell input, was reported to be higher
among obese compared with metabolically healthy over-
weight and lean persons, and to correlate with BMI,
adiposity measures (waist and WHR) and the degree of
insulin resistance (HOMA-IR) (25). Mechanistically, in
vitro insulin induced a signicant decrease in Th1\Th2 ratio
in lymphocytes from metabolically healthy overweight
persons, but not in insulin resistant obese persons (25,87).
Thus, insulin resistance may occur at the level of CD4+ T
cells, providing a link between obesity and disturbed Th1/
Th2 balance. Supporting this notion was an intervention
study demonstrating that weight loss achieved by VLCD
followed by surgery or VLCD only in morbidly obese men
caused a decrease in Th1\Th2 ratio, the degree of which
correlated with the extent of weight loss and decline in
waist circumference (81).
T cells subpopulations: circulating T regs (Table 3, iv).
Tregs, previously known as suppressor T cells, are anti-
inammatory cells most known for their role in allergies
and peripheral immune tolerance (88). While recent studies
suggest that AT Tregs may be decreased in obesity and
replenishing AT Tregs may improve insulin sensitivity
(reviewed in (89) ), in humans reports both on AT
and on circulating Tregs are inconsistent (3). Circulating
Treg cell counts (both nave, memory and natural
CD4
+
CD25
+
Foxp3
+
) were reported to increase in morbidly
obese adults and to correlate with BMI (76), although
others (90) reported that the percentage of Treg cells from
total CD4+ T cells was lower in obese compared with lean
(median: 1.2 and 0.73%, respectively), and correlated
negatively with BMI and body weight. Yet, in children no
difference was found among weight groups (86). According
to some studies, Tregs percentage might constitute an indi-
cator of a persons metabolic state: Treg cells percentage
was inversely correlated with leptin levels and glucose tol-
erance (HbA1c), and having Tregs < 1.06% was associated
with a 9.6-fold higher risk of having an inammatory obese
phenotype (hsCRP > 3.0 mg L
1
), and a lower level of
Tregs associated with higher HbA1c (90). In the same
study, Treg cell counts did not show many of the mentioned
correlation in the non-obese subgroup (90). Consistently, in
children, a lower percentage of Treg cells (identied as
CD4
+
CD25
high
CD127
low
FoxP3
+
PBMC) was observed in
obese children with the MetS compared with lean children
(85). One explanation might be a decreased differentiation
capacity towards Treg cells in a dysmetabolic state, as was
demonstrated in vitro in isolated CD4
+
CD25

cells from
obese children with the MetS compared with lean children
(85). From these studies it seems that Treg cells proportion
of CD4+ T cells and/or their function or inducibility, rather
than their absolute total number, may be more related to
obesity-associated dysmetabolism.
Early reports on impaired T cell immunity response in
mice with defective leptin (ob/ob) (91) or its receptor (db/
db) (92), together with studies, which demonstrated spe-
cic effects of leptin on T-lymphocyte responses (93,94),
suggest a crucial role for this adipocytokine as a mediator
between nutritional state/adiposity and cellular immune
function. Leptin receptor is present in T lymphocytes and
affects their activation (95), and particular attention has
been given to its role in Tregs cells, in which leptin seems
to affect their generation and proliferation both in mice
and humans (88). Yet, while it was recently reported that
decreased Treg percentage in the circulation correlates
with leptin (90), others did not observe clear associations
between T lymphocytes or T lymphocytes subpopulations
and leptin in adults (96) or children (97). Nevertheless, the
potential roles of adipokines (leptin, adiponectin) and
major factors regulating insulin sensitivity in obesity (such
as peroxisome proliferator activated receptor ) also in
Tregs biology provides multiple putative mechanisms for
links between the metabolic state and Treg-modulated
inammatory tone (89). Whether this can be estimated
from circulating Tregs remains to be proven.
Circulating B cells
B cells have been recently identied in mice and human
subcutaneous AT (98), and their role as contributors to the
development of AT insulin resistance has been demon-
strated in murine models (99). B cell counts (22,77) were
increased in obese (22,77,78) and in overweight (22) com-
pared with lean persons, and correlated with BMI, most
obesity reviews Circulating leucocytes and obesity subphenotypes T. Pecht et al. 11
2013 The Authors
obesity reviews 2013 International Association for the Study of Obesity
evidently in women (22). Additionally, in a large study
among men, B cell counts associated with an elevated risk
(odds ratio = 1.82) for MetS, the total number, and several
specic components of MetS (e.g. obesity, hypertension,
high TGs and low HDL) (78). In children, increased B
cell counts and percentage (by 1.5-fold) associated with
obesity-related liver disease as compared with children
with isolated obesity (97). Still, others reported that B cells
counts and percentage of total WBC did not correlate with
BMI in obese adults (62), and that total B cell counts did
not differ between obese children (79) and lean controls.
Circulating NK cells
In a community-based study comparing common-variety
obese and non-obese women, NK cell counts were not
signicantly different between the groups (although other
leucocyte classes were elevated in the obese women) (77).
Yet, in severely obese patients compared with lean controls
CD56+ NK cells were less abundant, accounting for
9.1 versus 12.3% of total peripheral lymphocytes (83).
Intriguingly, this could be largely attributed to obese
subphenotype associated with comorbidities: Comparing
26 unhealthy obese persons to 26 age-, BMI- and sex-
matched metabolically healthy obese, the former had
nearly 50% less circulating NK cells. Seemingly opposite
association between obesity-associated morbidities and NK
counts were reported in children (97): obese children in
whom obesity was accompanied by liver steatosis had on
average 66% higher absolute circulating NK cell counts
(dened as CD3/CD16+/CD56+ cells) compared with
children with isolated obesity. The difference between the
groups in absolute NK counts (although not their percent
of total lymphocytes) was the most signicant of all leuco-
cyte classes measured (97).
NK function is tightly regulated by diverse positive and
negative factors, including nutrients and metabolites. This,
along with non-standardized methodologies, likely underlie
inconsistencies in the literature. NKactivity was reported to
be unaltered in common-obesity (even when other leucocyte
functions were affected) (77), but decreased in severely
obese patients compared with lean controls (100). Further-
more, beyond constitutive (un-stimulated) cytotoxic activity
of PBMC (which is attributed to NK cells), NK seemed to
showaltered response to regulators. Adding to the complex-
ity, in the study mentioned above comparing metabolically
healthy to non-healthy obese, surface activation markers
revealed a complex dys-regulated state of NK activation: in
the unhealthy obese both inhibitory markers (NKB1 and
CD158b) and activatory markers (CD69) were expressed on
1218% of circulating NK cells (two- to threefold higher
expression than in healthy-obese, who were similar to lean
controls) (83). Cortisol-mediated inhibition of cytotoxicity
was diminished in obese compared with lean controls, an
effect largely seen in women, and suggested to represent a
leptin-mediated effect, such as by lowering the expression of
the glucocorticoid receptor (101). IL2-mediated activation
of cytotoxicity correlated with dietary carbohydrate content
and low-density lipoprotein levels, and negatively with
dietary lipid content (101).
Overall in longitudinal/intervention studies, diet-induced
weight loss, diet + exercise, or surgery-induced weight loss
did not signicantly alter NK counts (72,100), even when
total leucocytes, lymphocytes and neutrophils were signi-
cantly decreased (72). Yet, NK cytotoxic function exhibited
highly variable results: it was markedly increased in >75%
of severely obese persons 6 months after RYGB, reaching
the activity level of lean controls (100). This was attributed
to signicant elevations in IL-18, IL-12 and IFN (100),
cytokines thought to regulate NK cytotoxic function.
Taking a different approach i.e. assessing the surface
expression of CD69, another study exhibited time-
dependent decrease in this early activation marker follow-
ing weight-loss surgery, with no change in their relative
abundance (102). In a non-surgical intervention study,
NK-attributed cytotoxicity of PBMC was decreased in
obese women after 8 weeks on a hypo-caloric diet, an effect
of diet that could be offset by also engaging in mild-
moderate exercise training (72).
In summary, because NK cells participate in innate
immune response to infection, but also in immune defence
against malignancy, interest in this specic leucocyte
subpopulation has been considerably increasing with the
recognition that obesity predisposes to increased risk of
several malignancies. Yet, this putative association between
obesity-related alterations in NK abundance and/or func-
tion and incident malignancy has not been directly tested so
far. Moreover, current literature reveals a complex picture
(Table 2), which seems to mainly indicate decrease in NK
numbers/functions in severely obese, but possibly not in
mildly obese patients. Moreover, multiple nutritional,
metabolic and hormonal factors may regulate NK function,
which overall may be diminished by hypocaloric diet in
mild-moderate obesity, but elevated in response to surgical
intervention in severely obese patients.
Conclusions and outstanding questions
While the causal role of systemic and adipose inammation
in obesity and its associated diseases may still be debated, it
is generally accepted that inammation occurring in the
context of obesity may signify a subphenotype that has
higher risk of cardio-metabolic morbidity. Circulating leu-
cocytes, being an integral part of the immune system and
the source of the majority of immune cells inltrating
adipose tissue in response to obesity, are obvious bio-
marker candidates for identifying the more morbid forms
of obesity. However, as mentioned earlier in this review,
using either total leucocyte counts or the main leucocyte
12 Circulating leucocytes and obesity subphenotypes T. Pecht et al. obesity reviews
2013 The Authors
obesity reviews 2013 International Association for the Study of Obesity
classes has so far fallen short from being clinically useful
in risk-stratication of the individual obese patient. As
reviewed herein, current literature, although frequently not
fully consistent (Tables 2,3), may highlight some potential
directions for future research that may bring us closer for
making the assessment of circulating WBCs more clinically
useful:
Continued search for specic leucocyte subclass: The
full repertoire of leucocyte subclasses has yet to be identi-
ed. Specic molecular markers are continuously being
discovered and proposed to signify unique subclasses,
which may prove to be more tightly associated with better-
dened obesity subphenotypes. This effort is challenged by
the need to standardize methodologies and nomenclature
that would enable merging results from studies done by
different laboratories and comparing various human popu-
lations. It is noteworthy that results are sometime reported
in absolute counts or in relative abundance (percent of the
entire subgroup or total leucocytes). As exemplied in the
case of CD4+ T cells, this non-standardized way of express-
ing the data may contribute to apparent inconsistencies in
the literature.
Functional characterization of circulating cells: Func-
tional assays may prove valuable, even beyond identica-
tion of a specic subpopulation that is based on molecular
signatures. Table 2 reveals some instances in which func-
tional assays provide more consistent results than cell
counts and/or percentages. Moreover, altered function may
more strongly shed light on the mechanisms tying a specic
cell type to the cardio-metabolic morbidity that accompa-
nies obesity.
Assessment of circulating cells vis--vis adipose tissue
inammation: Chronic inammation of adipose tissue
does seem to characterize the more morbid obesity
subphenotypes (7). Yet, studies attempting to link a specic
circulating leucocyte population to adipose tissue inam-
mation are scarce, and in the case of T-lymphocyte sub-
classes unavailable (Table 3). Since adipose tissue,
particularly visceral fat, is usually not available for exami-
nation, identifying circulating blood cells that will signify
the state of intra-abdominal fat inammation would be
potentially very useful clinically.
Longitudinal and intervention trials: As can be noted
by Tables 2 and 3, the vast majority of existing literature
is cross-sectional. While providing good starting points,
the added insight provided by the few prospective and
interventional studies, especially when large-scale, is
unmatched. Yet, large data-sets would be required to
provide the most valuable information: assessing whether
circulating leucocyte subclass(es) could reveal obesity-
associated cardio-metabolic risk beyond already estab-
lished parameters, like the presence of the MetS. Strong
predictive relationship between leucocytes and incident
morbidity would inspire also mechanistic studies. It is
hoped that such studies could ultimately yield novel
approaches to relieve, in susceptible patients, the excess
cardio-metabolic risk they encounter by being obese.
Acknowledgements
TP was supported by a graduate students fellowship from
the National Institute of Biotechnology in the Negev,
Ben-Gurion University of the Negev, Beer-Sheva, Israel.
This work was supported by a grant from Deutsche
Forschungsgemeinschaft (DFG): SFB 1052/1: Obesity
mechanisms (project B02, to A.R.). A.R. is Chair of the
Fraida Foundation in Diabetes Research.
Conict of interest statement
No conict of interest was declared.
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