obesity reviews

doi: 10.1111/obr.12133

Etiology and Pathophysiology

Peripheral blood leucocyte subclasses as potential biomarkers of adipose tissue inflammation and obesity subphenotypes in humans T. Pecht1,2, A. Gutman-Tirosh1, N. Bashan1 and A. Rudich1,2

1

Department of Clinical Biochemistry and

Pharmacology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel; 2The 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: [email protected]

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 association, forming a unique obese subphenotype(s). Current approaches to define such subphenotypes include clinical biomarkers that largely reflect 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 inflammation, 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 inflammatory markers (C-reactive protein), adipokines (retinolbinding protein 4, adiponectin) and cytokines have been shown to correlate in humans with adipose tissue inflammation 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 infiltrating adipose tissue immune cells in obesity. In the present review, we summarize currently available literature on ‘classical’ blood white cell classes and on more specific 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 inflammation. Keywords: Cardio-metabolic risk, circulating leucocyte classes, metabolic syndrome, peripheral blood immune cells. obesity reviews (2014) 15, 322–337

Introduction Low-grade systemic inflammation is now well-accepted to accompany human obesity, and is thought to aetiologically contribute to obesity-associated cardio-metabolic comorbidities. The precise aetiology and relative contribution of obesity-associated systemic inflammation 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 inflammatory processes involving 322 15, 322–337, April 2014

immune cells within specific 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 inflammation, and may therefore serve to conceptualize the relations between specific organ and systemic inflammation in obesity. A detailed description of the evolution of the ‘adipose tissue inflammation concept’ in obesity is beyond the scope of the present review. Yet, it seems fair to say that adipose tissue inflammation obeys major paradigms of immune response (1),

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such as the early involvement of neutrophils (2), the subsequent recruitment of various lymphocyte populations (3) and the ultimate recruitment and polarization of macrophages, as well as mast cells (4). Moreover, adipose inflammation in obesity may undergo dynamic transitions from classical pro-inflammatory cascades to inflammationresolution and tissue remodelling processes (5,6). The impact of systemic and adipose inflammation in human obesity, in particular intra-abdominal fat inflammation, is exemplified by the highly intriguing subphenotype of the insulin-sensitive (or ‘metabolically healthy’) obese (7). This human obese subphenotype seems to escape the welldocumented associations between increasing body mass index (BMI), systemic inflammation, and cardio-metabolic morbidity. Importantly, insulin-sensitive obese persons are frequently characterized by a relative absence of intraabdominal fat inflammation compared with BMI-matched persons (7,8). However, because intra-abdominal fat is not readily available for clinical assessment, and because adipose-infiltrating immune cells in obesity are largely derived from bone marrows, a major gap of knowledge is whether specific circulating leucocyte (sub)populations could serve as markers of intra-abdominal fat inflammation, systemic inflammation, and thereby, of obesityassociated 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, particularly on the expanding role of inflammation, this finding 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 background, education level, height, smoking (active and passive) and fitness level. Second, the range of WBC counts typically varies by no more than 20% among persons stratified 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 significant overlap between the comparison groups. This complicates the ability to define clinically meaningful cut-off values. Third, despite a large body of data, even today it is still difficult 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 incident diabetes, deterioration of peripheral insulin sensitivity, metabolic syndrome (MetS) © 2013 The Authors obesity reviews © 2013 International Association for the Study of Obesity

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or coronary artery disease (13–16). Yet, a more clinically significant 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 specific leucocyte classes. Information on the major leucocyte classes (monocytes, lymphocytes, granulocytes) was greatly facilitated by automated differential blood counts technology. However, even with those major classes of circulating leucocytes, 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 inflammation and immunity in cardio-metabolic diseases, as well as on subclasses of circulating blood leucocytes within each of its major groups. These are defined by a growing number (although frequently still debated) specific and semi-specific surface markers that can be used to recognize, quantitate and isolate such cell subpopulations by robust technologies, as fluorescenceassisted 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 specified subpopulations of circulating blood leucocytes in cardio-metabolic diseases. Hopefully, results of such efforts will complement findings over recent years, in which cytokines levels have dominated the assessment of inflammatory tone and its potential contribution to disease.

Literature search approach Studies included in this review were those addressing the potential of different leucocytes populations/subpopulations, as defined in Table 1 and Fig. 1, to associate with human obesity in clinical cross-sectional and longitudinal studies (Tables 2 and 3). More specifically, in crosssectional 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 definition of

Table 1 The major classes and subclasses of circulating leucocytes discussed in this Review Circulating cell type

Normal count (count per mm3) (103)

Monocytes Neutrophils Eosinophils Basophils Lymphocytes

300–600 3,000–6,000 150–300 0–100 1,500–4,000

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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).

the MetS, and healthy/insulin sensitive obese versus insulinresistant obese. (iii) Is a specific leucocyte class associated specifically with adipose tissue inflammation? For longitudinal 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) infiltration 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 macrophages). Higher mean monocyte counts, typically by 13–18%, were reported in overweight and obese compared with lean persons (21), to correlate with BMI (22) and with

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higher prevalence of the MetS (16). Yet, others did not find such correlation (23,24), and even reports of negative association between the percentage of total monocytes and BMI could be found (25). Nevertheless, potential confounders were also identified in some studies: monocyte counts correlated with BMI only in women, but not in men (22), in whom, in another study, adjusting for fitness level abolished the crude correlation between monocytes and BMI (26). Monocytes’ gene expression and expression of specific surface molecules were demonstrated to differ between obese and non-obese persons, and to correlate with obesityrelated clinical abnormalities: a higher (approximately twofold) activated state (integrin CD11b surface expression) was noted in obese compared with non-obese, and the level of CD11b expression correlated with insulin resistance (higher homeostatic model assessment – insulin resistance [HOMA-IR] ) (25). Following the notion that ATMs in obesity may undergo a phenotypic switch from ‘alternatively activated’ resident macrophages (M2) to ‘classically activated’, pro-inflammatory macrophages (M1) (27), M1\M2 terminology has also been adopted to characterize

© 2013 The Authors obesity reviews © 2013 International Association for the Study of Obesity

© 2013 The Authors obesity reviews © 2013 International Association for the Study of Obesity

Yes (31): (34)* (36,38)* (41)

(37)

iii. Follow up/intervention longitudinal studies: is there effect of weight loss?

iv. Correlate with adipose tissue inflammation

*n > 100; **n > 1,000.

(+) (23):* (25) (28)* (31) (34)* (38)* (0) (24):*

(+) (23,36,38):* (39)* (40)* (37)

Association with a defined subpoplulation

ii. Association with obesity subphenotypes, like MetS and insulin resistance (cross-sectional [CS])

(+) (25):* (28)* (29–31,35)

Association with functional or activation markers

(0) (24):*

(−) (31):

(0) (22):* (25,79,80)

(+) (22):* (46) (76) (77)*

T cells

Yes (16):** (11)* (47)* (49)

(+) (11):* (44)* (45)* (0) (24):* (0) (16):** (24)* (63)*

No (36):

Yes (11):* (16)* (80) (72)

(+) (16):** (11)* (24)* (61)**

(see next & Table 3)

No (80):

Yes (31,81):

(+) (24):* (73) (78)**

(see Table 3)

(−) (31):

(−) (77):* (0) (73):

(+) (11):* (16)** (60)** (61)* (71)** (0) (21):* (22)* (46)

Total lymphocytes

(−) (36,53):*

(+) (61):**

(0) (21):* (26)* (60)** (62)* (63)*

(+) (36):

Eosinophils

(+) (22):*

(−) (64):*

(0) (16):** (21)* (62)* (63)*

(+) (26):* (60)** (61)**

Basophils

(+) (49,52,53):

(0) (21):*

(0) (23):* (62)*

(−) (25):

(+) (11):* (44)* (46)

(+) (16):** (21)* (22)* (26)*

i. Association of leucocyte number or % with body mass index/adiposity (cross-sectional [CS] )

Neutrophils

Monocytes

Level of association with obesity

Table 2 Summary of studies assessing major classes of circulating leucocytes in human obesity

(+) (78):** (97)

(0) (62):* (79)

(+) (22):* (77)* (78)**

B cells

Yes (72,100,102): function No (72,100):

(−) (83):

(+) (97):

(−) (83,100): (0) (77):*

(+) (83,101):

(−) (83):

(0) (62):* (77)* (102)

NK cells

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circulating monocytes. The underlying notion was perhaps that ATM subpopulations are derived from ‘predetermined’ specific circulating monocytes. Higher expression of the M1 markers tumour necrosis factor α (TNFα) and interleukin (IL-6), and lower expression of the M2 marker IL-10 were found in monocytes from obese nondiabetic persons compared with lean controls (28). Additionally, 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 obesityassociated morbidity (28). Further following this notion, in a multivariate regression analysis, the mRNA expression of the M1-related markers TNFα and IL-6 was associated with BMI, while IL-6 also associated with low-density lipoprotein 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 differentiation into an M2 phenotype (lower M2 gene expression) in both diabetic and non-diabetic obese compared with lean women (29). This latter finding suggests that ATM polarization might be already programmed in the circulating monocytic precursor cell, a notion also supported by functional in vitro assays: Increased reactive oxygen species (ROS) and activation of endoplasmic reticulum stress were demonstrated in circulating monocytes from obese nondiabetic compared with lean persons (30); peripheral blood mononuclear cell (PBMC)-derived monocytes from obese persons with no comorbidities exhibited significantly increased basal and lipopolysaccharide (LPS)induced TNFα production compared with non-obese, correlating with metabolic parameters, TG levels 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 controls was monocytes’ insulin-induced receptor internalization (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.

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The mechanisms for the possible circulating monocytes’ programming in obesity and obesity-associated diabetes is a growing area of interest: micro-RNAs have been implicated in the deregulation (elevated expression) of some M1 markers in circulating monocytes in obesity: miR-181a, −181b and −181d were identified as possible regulators of the toll-like receptor (TLR)/nuclear factor kappa-lightchain-enhancer of activated B cells (NFκB) signalling potentially by targeting TLR4, the NFκB adapter molecules TRAM, TAK1 and TAB2, and the inflammatory cytokine TNFα. The expression level of these miR’s in CD14+ PBMC (considered as monocytes) negatively correlated with systemic inflammation (circulating IL-6 and highsensitivity 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 angiography exhibited lower levels of miR-181a, but not of miR181b and miR-181d. This de-regulated expression of miR181a 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 identified to decrease in monocytes from obese compared with lean persons was miR-146b-5p, which suppresses the IL-1 receptorassociated kinase (IRAK)/NFκB pathway adapter molecules IRAK1 and TNF receptor-associated factor-6 (TRAF6) (35). Specific 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-affinity FcγIII receptor. Three human monocyte subpopulations have been recently defined based on these surface markers: CD14+CD16− (‘classical monocytes’), CD14+CD16+ (‘intermediate monocytes’), and CD14dimCD16+ (‘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 correlate 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 attenuated when adjusting for the CD16+ monocytes count, suggesting that the increase in CD16+ monocyte counts is in the same causal pathway linking obesity to subclinical atherosclerosis (23). Importantly, circulating CD16+ monocytes correlated with subcutaneous ATMs (CD68+ cells), © 2013 The Authors obesity reviews © 2013 International Association for the Study of Obesity

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Circulating leucocytes and obesity subphenotypes T. Pecht et al.

both of which were elevated in a cohort of normoglycaemic obese versus lean controls (37), indicating that specific monocyte subclass(es) may constitute a marker for adipose tissue inflammation, and thus potentially, for the tendency to develop a more cardio-metabolic morbid form of obesity. When considered separately, CD14+CD16+ and CD14dimCD16+ were both increased, by 70–100%, in obese compared with lean persons, and showed an association with glucose tolerance, insulin sensitivity (glycaemia, insulin, HOMA-IR), inflammation markers (hsCRP) and adiposity (BMI and fat mass) (38). Yet, CD14+CD16+ (intermediate) monocyte subpopulation did not correlate with BMI (23), whereas CD14dimCD16+ (alternative) monocytes were higher in obese diabetic compared with non-diabetic (38), and their counts correlated with parameters 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 inconsistent 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) monocyte 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 verylow-energy diet (VLED) intervention was accompanied by a decrease in monocyte counts in normoglycaemic obese persons compared with baseline (41). Moreover, microRNA (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 TNFα production by PBMCderived monocytes was lower in obese persons with no comorbidities after VLED compared with baseline (although remained elevated compared with non-obese persons) (31). Similar findings were reported with specific 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). © 2013 The Authors obesity reviews © 2013 International Association for the Study of Obesity

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Interestingly, a decrease specifically in the CD14dimCD16+ 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 finding in some studies involving smaller number of participants (Table 2). Yet, several specific 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 intervention. An additional possible means of increasing the potential significance of monocyte measurements is by assessing specific 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 inflammation.

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 infiltrate 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 inflammation (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 association of BMI and adiposity with increased neutrophil counts, with the highest correlation coefficients compared with any other leucocyte population assessed (46). In Byukkaya et al. (45), 70 MetS (not necessarily obese) persons exhibited significantly 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 finding – see section dealing with lymphocytes). Putting

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these somewhat unique characteristics of this cohort aside, a neutrophil-to-lymphocyte (NLR) ratio of >1.84 (suggested to represent a high ‘pro-inflammatory load’) had a sensitivity (true-positive rate) of 92.8% and a specificity (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 lifestyle (diet ± exercise) (47) and surgical interventions (11) were reported to decrease neutrophil counts. A greater 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). Interestingly, in post-menopausal women randomized for 1 year of either hypocaloric diet alone, exercise, or their combination, diet (and not exercise) was the driver for a decline in neutrophil counts (and other inflammatory 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 reflect, beyond generic issues such as the specific patients’ characteristics, factors more specific to neutrophils’ biology and to how it is studied: neutrophil activation is a multifaceted process that involves multiple stages (priming versus activation) and specific 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 methodologies used to estimate specific 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

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surgery-induced weight loss. Contrasting the view of diminished neutrophil function in obesity, surface expression of neutrophil CD66b, considered a marker of neutrophil activation (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 products myeloperoxidase and calprotectin, the second of which significantly 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 formyl–methionyl–leucyl–phenylalanine-induced IL-8 (CXCL8) production by neutrophils were increased in overweight 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 diminished bacterial ingestion capacity, and stimulus-activated ROS production, with no apparent effect of obesity on chemotaxis. Impaired phorbol12-myristate-13-acetateinduced 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 significantly elevated in morbidly obese compared with controls (53). Moreover, 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). Collectively, it would seem that obesity associates with a complex alteration of neutrophils’ inflammatory function, potentially with partial, basal, pro-inflammatory activation and possibly diminished ability to fully activate proinflammatory 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 specifically for neutrophils are adiponectin and lactoferrin. The obesity-associated decrease in the adipocyte-derived hormone adiponectin was proposed to sensitize neutrophils to stimulus-induced CXCL8 secretion in obesity (52). Interestingly, lactoferrin, another protein with antiinflammatory effects, was found to be mildly decreased in persons with dysglycaemia/diabetes, both at the circulating © 2013 The Authors obesity reviews © 2013 International Association for the Study of Obesity

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level and ex vivo in LPS-stimulated whole blood (56). Both factors may link obesity with neutrophils’ pro- inflammatory 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 association with basophils and eosinophils that are inherent components of allergic inflammation. Both of these populations 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 inflammation is limited and rather inconsistent. Basophils play an important role in the initiation and propagation of immediate-type hypersensitivity reactions by binding antigen-specific immunoglobulin E (IgE) that triggers their activation, granule exocytosis, and the release of histamine and other allergic-reaction mediators. Currently there are no reports on increased presence of basophils in AT. Nevertheless, leptin receptor on human basophils was found to mediate enhanced survival, migration, 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, education 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 crosssectional 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 tomography) was reported in adolescent women after adjusting for age, blood pressure, total cholesterol, HDL-cholesterol, TG and fasting glucose (64). © 2013 The Authors obesity reviews © 2013 International Association for the Study of Obesity

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Beyond peripheral basophil counts, serum concentration 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 specific 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 specific IgE and BMI in young adults (68). Eosinophils have been proposed to regulate adipose tissue inflammation by maintaining ATMs in an alternative activation (M2) state via IL-4 production (69). Yet, as with basophils, there are conflicting 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 findings 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 differential WBC counts, and can be readily isolated from whole blood. Hence, we first briefly review lymphocytes’ relevance in obesity as a single group, followed by consideration of specific 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 associated 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 crosssectional analyses, in the large longitudinal PREDIMED study, a multivariate logistic regression analysis (although not Cox regression) revealed that persons classified in the top quartile of lymphocyte counts at baseline (i.e.

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2.55 × 109 cells per L versus 1.54 × 109 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). Additionally, 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 baseline, and those with a greater percentage of weight reduction (11). Despite the highly suggestive studies mentioned earlier for a positive association between total circulating lymphocytes 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 production of inflammatory cytokines by lymphocytes was similar in overweight and lean young adults (73). T cells T lymphocytes, commonly identified by expressing CD3, have been recently implicated in obesity-related AT inflammation (1). They were reported to infiltrate 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 conflicting reports: Total T cell counts were found to increase by 15–50% 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+ T cell counts may be linked to the metabolic state. Higher CD3+ T cell counts were demonstrated to associate with MetS and with specific 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

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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 metabolically healthy overweight, compared with lean persons (25). Yet, expression of both CD25 and CD69 decreased following VLCD + surgery-induced weight loss, with CD25 dropping by more than threefold, comparable with lean controls (81). Using a different T cell activation marker, HLA-DR (MHC class 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 conflicting data about associations between circulating total T cell counts and obesity and/or its comorbidities may reflect, 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 represent the diversity of T cell subpopulations. Here, we will first review current information on obesity and circulating cytotoxic T cells (CTL), cells characterized by the expression of CD8, a co-receptor of the T-cell receptor, which mediates efficient cell–cell interactions; we will then discuss jointly the CD4+ T cell subpopulations, followed by separate 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 considered to promote pro-inflammatory response, Th2 and Treg are thought to suppress or limit it. Subsequent sections further discuss how these specific T cell lymphocyte subpopulations in circulating blood associate with obesity, comorbidity and AT inflammation. T cells subpopulations: circulating CD8+ CTL (Table 3, i). Cytotoxic CD8+ T cells were suggested to infiltrate adipose tissue in both mice and humans, and to promote the (subsequent) recruitment, differentiation and activation of macrophages (3). In the circulation in humans, 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 subphenotypes 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 © 2013 The Authors obesity reviews © 2013 International Association for the Study of Obesity

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Table 3 Summary of studies assessing circulating T cell subclasses in human obesity Level of association with obesity

i. Cytotoxic T cells (CD8+)

ii. CD4+

iii. Th1,Th2,Th1/Th2

iv. Tregs

i. Association of leucocyte number or % with body mass index/adiposity (CS)

(+) (84): (0) (77): (−) (54,73,79,80,82,83):

(+) (22,46,60,62,76,80,82,84): (0) (22,62,73,83,85,86): (−) (31):

(+) (25,76,79): (0) (76):

(+) (76): (0) (86,90): (−) (90): (%of CD4+)

Association with functional or activation markers

(+) (76):

(+) (76):

ii. Association with obesity subphenotypes, like MetS and insulin resistance (CS)

(+) (73): (−) (83):

(+) (76,78):

(+) (25,76,79):

(85) (90)

(0) (31,81):

(0) (80,81): (−) (31):

iii. Follow up/intervention longitudinal studies

(−) (85):

(+) (81):

iv. Correlate with adipose tissue inflammation

counts associated with the presence of the MetS, correlating with the number of positive criteria for the MetS, specifically 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 significant causal role in obesity-associated cardiometabolic morbidity, or at least in improvements in risk factors following weight-loss interventions. T cells subpopulations: circulating CD4+ (Table 3, ii). Circulating 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 correlate with BMI (76,82). CD4+ T cells also showed correlations 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, specific 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 specific dominant antigen(s), as the T cell receptor β repertoire was diverse. In contrast, CD4+ T cell blastogenic capacity was lower in obese © 2013 The Authors obesity reviews © 2013 International Association for the Study of Obesity

compared with non-obese persons, and was increased after VLCD-induced weight loss (31). It is worth noting that unlike the fairly consistent crosssectional 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 correlation 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 unaltered between obese and formerly obese women who (historically) underwent bariatric surgery and nutritional intervention (80). Even more directly, CD4+ T cell percentage 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-inflammatory immune response, involving secretion of cytokines such as interferon-γ (IFNγ) and participation in cell-mediated immune responses, while Th2 cells are largely antiinflammatory cells, producing IL-4 and IL-13 and promoting the humoural immune response (4). In AT, the main paradigm states that Th1 cells increase with obesity, while Th2 (and Treg’s, see next section) decrease, shifting the delicate balance between pro- and anti-inflammatory immune cells within the tissue towards a more proinflammatory tone (3). To our knowledge, in the circulation,

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Th1 and Th2 absolute counts have not been frequently reported, although more information is available on their relative abundance (percentage of CD4+ T cells). Th1 counts (identified as CD4+IFNγ+ PBMC cells) 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 (identified as CD4+ IFNγ 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 nonalcoholic 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 (identified as CD4+IL-4+ PBMC cells) were reported to increase in morbidly obese persons and to correlate with BMI (76), although no difference 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-inflammatory T cell input, was reported to be higher among obese compared with metabolically healthy overweight 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 significant 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 antiinflammatory cells most known for their role in allergies and peripheral immune tolerance (88). While recent studies suggest that AT Treg’s 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 naïve, 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

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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 indicator of a person’s metabolic state: Treg cells percentage was inversely correlated with leptin levels and glucose tolerance (HbA1c), and having Tregs < 1.06% was associated with a 9.6-fold higher risk of having an inflammatory 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 (identified as CD4+CD25highCD127lowFoxP3+ 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 specific 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 Treg’s 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 γ) also in Treg’s biology provides multiple putative mechanisms for links between the metabolic state and Treg-modulated inflammatory tone (89). Whether this can be estimated from circulating Tregs remains to be proven. Circulating B cells B cells have been recently identified in mice and human subcutaneous AT (98), and their role as contributors to the development of AT insulin resistance has been demonstrated in murine models (99). B cell counts (22,77) were increased in obese (22,77,78) and in overweight (22) compared with lean persons, and correlated with BMI, most © 2013 The Authors obesity reviews © 2013 International Association for the Study of Obesity

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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 specific 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 significantly 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 sexmatched 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 (defined 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 significant of all leucocyte 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. NK activity 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). Furthermore, beyond constitutive (un-stimulated) cytotoxic activity of PBMC (which is attributed to NK cells), NK seemed to show altered response to regulators. Adding to the complexity, 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 12–18% 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 © 2013 The Authors obesity reviews © 2013 International Association for the Study of Obesity

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leptin-mediated effect, such as by lowering the expression of the glucocorticoid receptor (101). IL–2-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 significantly alter NK counts (72,100), even when total leucocytes, lymphocytes and neutrophils were significantly 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 significant elevations in IL-18, IL-12 and IFNγ (100), cytokines thought to regulate NK cytotoxic function. Taking a different approach – i.e. assessing the surface expression of CD69, another study exhibited timedependent decrease in this early activation marker following 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 mildmoderate 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 specific 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 function 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 inflammation in obesity and its associated diseases may still be debated, it is generally accepted that inflammation occurring in the context of obesity may signify a subphenotype that has higher risk of cardio-metabolic morbidity. Circulating leucocytes, being an integral part of the immune system and the source of the majority of immune cells infiltrating adipose tissue in response to obesity, are obvious biomarker 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

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classes has so far fallen short from being clinically useful in risk-stratification 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 specific leucocyte subclass: The full repertoire of leucocyte subclasses has yet to be identified. Specific molecular markers are continuously being discovered and proposed to signify unique subclasses, which may prove to be more tightly associated with betterdefined 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 populations. It is noteworthy that results are sometime reported in absolute counts or in relative abundance (percent of the entire subgroup or total leucocytes). As exemplified in the case of CD4+ T cells, this non-standardized way of expressing the data may contribute to apparent inconsistencies in the literature. • Functional characterization of circulating cells: Functional assays may prove valuable, even beyond identification of a specific subpopulation that is based on molecular signatures. Table 2 reveals some instances in which functional assays provide more consistent results than cell counts and/or percentages. Moreover, altered function may more strongly shed light on the mechanisms tying a specific cell type to the cardio-metabolic morbidity that accompanies obesity. • Assessment of circulating cells vis-à-vis adipose tissue inflammation: Chronic inflammation of adipose tissue does seem to characterize the more morbid obesity subphenotypes (7). Yet, studies attempting to link a specific circulating leucocyte population to adipose tissue inflammation are scarce, and in the case of T-lymphocyte subclasses – unavailable (Table 3). Since adipose tissue, particularly visceral fat, is usually not available for examination, identifying circulating blood cells that will signify the state of intra-abdominal fat inflammation 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 obesityassociated cardio-metabolic risk beyond already established parameters, like the presence of the MetS. Strong predictive relationship between leucocytes and incident

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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 student’s 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.

Conflict of interest statement No conflict of interest was declared.

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