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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
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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