Analysis of the percentages of monocyte subsets and ILC2s, their relationships with metabolic variables and response to hypocaloric restriction in obesity

Nicté Figueroa-Vega; Carolina I Marín-Aragón; Itzel López-Aguilar; Lorena Ibarra-Reynoso; Elva Pérez-Luque; Juan Manuel Malacara

doi:10.1371/journal.pone.0228637

. 2020 Feb 19;15(2):e0228637. doi: 10.1371/journal.pone.0228637

Analysis of the percentages of monocyte subsets and ILC2s, their relationships with metabolic variables and response to hypocaloric restriction in obesity

Nicté Figueroa-Vega ^1,^*, Carolina I Marín-Aragón ¹, Itzel López-Aguilar ¹, Lorena Ibarra-Reynoso ¹, Elva Pérez-Luque ¹, Juan Manuel Malacara ^1,^*

Editor: Melissa M Markofski²

PMCID: PMC7029876 PMID: 32074122

Abstract

Purpose

Obesity results from excess energy intake over expenditure and is characterized by chronic low-grade inflammation involving circulating monocytes (Mo) and group 2 innate lymphoid cells (ILC2s) imbalance. We analyzed circulating Mo subsets and ILC2s percentages and β2-adrenergic receptor (β2AR) expression in lean and obese subjects, and the possible effect of hypocaloric restriction on these innate immune cells.

Methods

In 139 individuals aged 45 to 57 years, classified in 74 lean individuals (>18.9kg/m² BMI <24.9kg/m²) and 65 with obesity (n = 65), we collected fasting blood samples to detect Mo subsets, ILC2s number, and β2AR expression by flow cytometry. Lipids, insulin, leptin, and acylated-ghrelin concentrations were quantified. Resting energy expenditure (REE) was estimated by indirect calorimetry. These measurements were repeated in obese subjects after 7-weeks of hypocaloric restriction.

Results

Non-classical monocytes (NCM) and β2AR expression on intermediate Mo (IM) were increased in obese individuals (p<0.001, in both cases), whereas the percent of ILC2s was decreased (p<0.0001). Stepwise regression analysis showed significantly negative associations of ILC2s with caloric intake, β2AR expression on IM with REE, but a positive relationship between NCM and HOMA-IR. Caloric restriction allowed a significant diminution of NCM and the β2AR expression on IM, as well as, an increase in the percent of classical Mo (CM), and ILC2s. ΔREE was related to ΔCD16⁺/CD16^- ratio.

Conclusions

These findings show that in obesity occur changes in NCM, ILC2s and β2AR expression, which contribute to the low-grade inflammation linked to obesity and might revert with caloric restriction.

Introduction

In obesity, the imbalance of energy intake/expenditure favors the accumulation of fat [1], usually with chronic low-grade inflammation [2], which has effects on energy metabolism at central and peripheral levels. Inflammation induces fat mobilization and oxidation [2]. Obese individuals are supposed to have lower energy expenditure (REE), however, longitudinal studies show REE increase with obesity [1].

Several immune cell types are key regulators of metabolic homeostasis [3–7]. In white adipose tissue (WAT) from obese subjects change immune cell composition and function with an effect on energy expenditure. Immune cells in the adipose tissue (AT) of lean subjects include T regulatory (Treg) cells, iNKT cells, group 2 innate lymphoid cells (ILC2s), and M2 macrophages; these cell types have distinct roles in the maintenance of AT homeostasis [2]. In obesity, excessive visceral fat accumulation causes adipose tissue dysfunction that leads to chronic-low grade inflammation with adipocyte hypertrophy and hyperplasia, shift from a type 2 to type 1 cytokine-associated inflammatory environment, altered secretion of adipokines (leptin, adiponectin, and other), and changes in proportions and kind of immune cells toward pro-inflammatory monocytes and Th17 lymphocytes, which strongly contributes to obesity-related comorbidities [3–5].

Adipokines are peptide mediators produced by fat cells in AT that exert a powerful influence over immune system [8]. Leptin and adiponectin have effect over functions of dendritic cells, monocytes, neutrophils, and innate lymphoid cells. Leptin, induces satiety, and with other metabolic functions such as regulation of energy expenditure. It also increases phagocytic activity, pro-inflammatory cytokines secretion, and polarization of immune cells toward pro-inflammatory phenotypes [9]. Appetite regulation has a counterregulatory peripheral function in ghrelin, an acylated peptide product of the stomach that induces appetite, and it is also an anti-inflammatory cytokine that suppresses inflammation in obesity [10].

Circulating monocytes (Mo) include three distinct subtypes according to their surface expression of lipopolysaccharide receptor CD14 and FcγIII receptor CD16 [11,12], as follows: classical monocytes (CM; CD14⁺⁺CD16^-) account for 80–90% of total monocytes with an anti-inflammatory phenotype. The minor CD16⁺ Mo subpopulation comprises the remaining 10–15% and is subdivided further into intermediate monocytes (IM; CD14⁺CD16⁺) and non-classical monocytes (NCM; CD14^-CD16⁺⁺), both with a pro-inflammatory phenotype and are elevated in chronic inflammatory and metabolic diseases [12–18]. Changes in monocytes subsets CD14⁺⁺CD16^-, CD14⁺CD16⁺ and CD14^-CD16⁺⁺ have been described after dietary interventions in obese individuals [19–22]. Monocytes can migrate toward AT and endothelium, where they become converted into macrophages. Therefore, it is important to detect these subpopulations in peripheral blood for the evaluation the chronic low-grade inflammation and regulation of energy in obesity.

Innate lymphoid cells (ILCs) comprise three subpopulations: ILC1, ILC2 and ILC3. Recent evidences indicate that ILCs are involved in the progression of several metabolic diseases. These cells promote obesity, and are involved in adipose tissue inflammation [23,24]. Nevertheless, group 2 innate lymphoid cells (ILC2s), anti-obese immune regulators in AT, secrete anti-inflammatory cytokines, promote polarization into M2 macrophages, eosinophil regulating adaptive immunity, limiting obesity and promoting the browning of WAT [25]. ILC2s synthesize IL-5 and IL-13, cytokines implicated in browning of WAT (thermogenesis) [26]. However, the mechanisms by which ILC2s regulate AT homeostasis are incompletely defined. ILCs has been characterized in AT but information about their presence in circulation is not available. Therefore, it is needed a new approach for their study and establish their connections with energy metabolism. Due to the complexity for the isolation of ILC2 cells from human AT, peripheral bloods specimens should be used to monitor ILC2s.

Sympathetic and parasympathetic systems converge in the activation of β2-adrenoceptors on immune cells to control systemic inflammation allowing the crosstalk between nervous, endocrine and immune systems [27]. β2AR are found in inflammatory cells such as mast cells, monocytes, eosinophils, T-lymphocytes, and neutrophils. β2AR expression on Mo and their activation has usually anti-inflammatory effect [28].

The aims of this work were to determine circulating Mo subsets and ILC2s and β2AR expression in obese and lean subjects, and to evaluate the effect of moderate diet restriction on these cells, and the interaction of resting energy expenditure (REE), and metabolic variables in subjects with obesity.

Materials and methods

Participants

We recruited 139 participants of 20 to 50 years old from León, Mexico, classified in two groups, 65 with obesity (BMI ≥30 (kg/m²), and 74 lean subjects (BMI >18.9kg/m² to ≤24.9kg/m²).

Participants did not have clinical evidence of chronic or infectious diseases and were not taking anxiolytics, antidepressants, β-blockers, Ca⁺⁺ channel blockers, antibiotics, or hypnotic drugs. Women did not receive hormone therapy in the previous six months, and were not pregnant, or lactating. No volunteer with smoking habit or habitual alcohol consumption was included.

Data collection

We collected age, weight measured with a roman type scale, and height with a Stadiometer (SECA 216). BMI and percentage weight loss (%WL) were calculated. Blood pressure was measured in sitting position after 5 min rest. Physical activity was evaluated using the International Physical Activity Questionnaire (IPAQ) [the short, last seven days self-administered version of IPAQ from the 2000/01 Reliability and Validity Study]. Collection of all measurements was performed in basal state and at the end of the diet period in individuals with obesity (Fig 1).

Fig 1 — Diagram illustrates the sequence of procedures carried out on the study for selection and classification of participants in both groups, assignation of diet in individuals with obesity, and measures.

Resting energy expenditure (REE)

REE was estimated by indirect calorimetry (IC) with a Fitmate device (Wellness Technology, Cosmed, USA), calibrated before each assessment, following the manufacturer’s specifications. For evaluation, subjects abstained from physical exercise, drinks with coffee or black tea the previous 24 h. The assessment was carried out after 8–10 h fasting in a controlled environment with room temperature 21–24°C, with low light and no noise. During the 15 minutes of measurement, subjects were awake and in supine position, measurements on the first 10 min were discarded to improve stability.

Samples

Peripheral blood samples were obtained after 8–10 h overnight fasting. Serum was separated and stored at -80°C until use. Peripheral mononuclear cells were isolated from heparinized blood. The samples were obtained before and after intervention for measurement of all variables.

Dietary intervention

Personalized caloric restriction was prescribed for 7-weeks, reducing 580 kcal of current intake of each individual with obesity. Dietary intake was designed in kilocalories/day with the following percentage of macronutrients: 55%–60% carbohydrates, 15% proteins and 30% lipids, as recommended for obese individuals, by the North American Association for the Study of Obesity (NAASO) and the National Heart, Lung, and Blood Institute (NHLBI) 2000.

Dietary intake was evaluated with 24-hour recollection (two weekdays and one weekend), at baseline and on each of the three weeks of the intervention. We analyzed 24-hours recollections with the Food Processor SQL-Nutrition Software, to quantify energy intake (Kcal) and type of macronutrients. Adherence to diet was considered satisfactory when energy consumption was ±80% of the amount prescribed. Normal weight subjects continued with their customary diet.

Metabolic and hormonal measurements

Serum glucose and lipid profile were measured using enzymatic methods with a chemical analyzer (Microlab 300, ELITECH group, Vital Scientific, Netherlands). LDL-cholesterol was calculated by the Friedewald´s formula [29]. Non-HDL cholesterol was also calculated. Circulating acylated-ghrelin (MyBiosource, CA, USA), insulin (ALPCO, Salem, NH), and leptin (ALPCO) concentrations were measured by ELISA. All analyses were carried out by duplicate.

Cell isolation

Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque (1.077 g/ml; Sigma-Aldrich) gradient. Viability was examined using trypan blue exclusion.

Identification of monocytes subsets

PBMC were stained with anti-CD14 mAb conjugated to Fluorescein isothiocyanate (FITC) and–CD16 mAb conjugated to Allophycocyanin (APC) (both purchased from BD Biosciences, San Jose, CA, USA). To set the gates, control isotype antibodies were employed, an anti-mouse Ig kappa chain-FITC (BD Pharmingen), and an anti-mouse IgG1-APC (BioLegend). Moreover, monocytes were gated on the basis of their size (forward scatter) and complexity (side scatter), and the monocyte subsets were identified by the levels of expression of CD14 and CD16. Fifty thousand events were acquired for each sample on a FACSCanto II (two-laser, six-color configuration) with the FACSDiva 6.1.3 software (BD Biosciences). The results are shown as percentage of each subpopulation. Nomenclature of monocyte subsets followed the recommendations of the Nomenclature Committee of the International Union of Immunological Societies. We carried out daily routine quality control tests with Cytometer Setup & Tracking Beads (BD Biosciences) in accordance with the manufacturer’s instructions.

Evaluation of β2AR expression

The level of expression of β2AR was analyzed by a three-color flow cytometry assay. Briefly, cells were incubated with an anti-β2AR (IgG1, clone 6H8; Abcam) mAb, followed by a goat anti-mouse IgG polyclonal Ab labeled with PE (Abcam). Then, cells were stained for CD14 and CD16, as stated above. Finally, cells (at least 50,000, and gated according to their FS and SS characteristics) were analyzed in a FACSCanto II (two-laser, six-color configuration) flow cytometer with the FACSDiva 6.1.3 software (BD Biosciences). Results were shown as the median intensity of fluorescence (MFI) of positive cells.

Phenotypic characterization of ILC2s

PBMC were stained with the following antibody mix: FITC-conjugated anti-human CD2, CD3, CD14, CD16, CD19, CD56, and CD235a (negative lineage cocktail), Phycoerytrin-(PE)-conjugated anti-CRTH2 (chemoattractat receptor-homologous molecule expressed on Th2 cells), Peridinin-Chlorophyll Protein Complex-(PerCP-)-Cyanin-5-conjugated anti-human CD127 (IL-7R) and Allophycocyanin-(APC-)-conjugated anti-human IL-5 (all purchased from BD) in presence or not of PMA/Io (Sigma-Aldrich, USA) and a Golgi inhibitor (Brefeldin A; BD Biosciences). Then, lymphocytes were gated according to their FSC/SSC characteristics, and lineage-negative (Lin^-) were selected and analyzed for the expression of CD127 and CRTH2. Finally, These Lin-CD127-CRTH2+ were analyzed for the intracellular expression of IL-5. According to this analysis, Lin^-CD127^-CRTH2⁺IL-5⁺ events were considered as ILC2 cells and cells were gated by running fluorescence minus one (FMO) control tubes. Cells were analyzed in a FACSCanto II (two-laser, six-color configuration) flow cytometer with FACSDiva 6.1.3 software (BD Biosciences).

Statistical analysis

Descriptive statistics was used to show the characteristics of subjects, normality was assessed with the Kolmogorov-Smirnov test. Data are shown as the means ± SD. We compared groups of lean vs obese subjects using the Student’s T test for independent variables. Changes in variables before and after diet were examined with paired Student´s T test.

We examined factors associated with monocyte subsets percent, ILC2s percent, and β2AR expression using multiple forward stepwise regression analysis testing as candidate regressors: REE, BMI, mean arterial tension, HDL-C, non-HDL-C, triglycerides, acylated-ghrelin, leptin, HOMA-IR, and caloric intake values; and as confounding factors: gender and age.

For analyses we used the Statistica 5.0 (Stat Soft Inc., Tulsa, OK), and Prism 7.0v (GraphPad) softwares. p<0.05 was considered statistically significant.

Ethical approval

All individuals gave written informed consent to participate in the study. The study was carried out according to the ethical standards of the Declaration of Helsinki in 1983 and in agreement with the Good Clinical Practice guidelines. The Institutional Ethics Committee of the University of Guanajuato approved the study with number CIBIUG No. 017/2015.

Results

We studied 65 unrelated obese subjects (34 women and 31 men) of 35.4±8.0 years old, and 74 lean subjects (51 women and 23 men) aged 30.1±7.0 years. No gender differences were observed between groups of subjects with and without obesity.

The comparison of characteristics among lean and obese subjects is shown in Table 1. REE was significantly higher in obese than lean subjects, but glucose and acylated ghrelin concentrations were not different between both groups. As expected, individuals with obesity had higher blood pressure and insulin resistance.

Table 1. Comparison of basal characteristics among individuals.

	Lean subjects Mean±S.D. (n = 74)	Obese subjects Mean±S.D. (n = 65)	t	p-value
Age (yr)	30.1 ± 7	35.4 ± 8	-4.0	<0.001
Weight (kg)	63.5 ± 9.3	89.5 ± 15.3	13.4	<0.0001
BMI (kg/m²)	22.8 ± 1.9	34.1 ± 4.7	19.1	<0.0001
Mean arterial tension (mmHg)	84 ± 7	90 ± 6.5	-5.6	<0.0000001
Resting energy expenditure (kcal/day)	1530 ± 286	1818 ± 380	-5.09	<0.0000011
Glucose (mg/dl)	89 ± 12	92 ± 13	-1.4	0.17
HDL-cholesterol (mg/dl)	62±11	52±12	5.1	<0.000001
Non-HDL-cholesterol (mg/dl)	112±32	140±40	-4.5	<0.000016
Triglycerides (mg/dl)	112±60	178±196	-2.8	<0.0067
Creatinine (mg/dl)	0.9±0.2	0.9±0.2	-0.6	0.52
HOMA-IR	1.9±0.8	3.6±2.2	-6.2	<0.00000001
Acylated-ghrelin (μg/ml)	125.3±100.4	104.0±47.6	1.4	0.16
Leptin (pg/ml)	21.7±18.2	43.4±24.5	-5.9	<0.00000002
Insulin (μUI/ml)	8.6±3.4	15.4±8.8	-6.2	<0.00000001
*Monocyte subsets*
Non-classical monocytes (NCM; CD14^-CD16⁺⁺) (%)	4.0±2.5	7.2±5.7	-4.4	<0.000022
Intermediate monocytes (IM; CD14⁺CD16⁺) (%)	6.50±4.3	7.4±5.0	-1.1	0.26
Classical Monocytes (CM; CD14⁺⁺CD16^-) (%)	65.0±11.7	62.7±15.1	1.0	0.31
CD16⁺/CD16^- ratio	0.2±0.1	0.3±0.2	-3.1	<0.0024
*Innate lymphoid cells*
ILC2s (%)	11.3±6.7	2.9±2.5	4.65	<0.0001
*β2AR expression within monocyte subsets*
β2AR expression on NCM (MFI)	10191±4738	11912±9718	-1.0	0.33
β2AR expression on IM (MFI)	25966±15911	28370±17169	-0.7	<0.001
β2AR expression on CM (MFI)	13363±5846	15877±12429	-1.1	0.50

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Data are shown as the means ± SD. Differences between groups were evaluated with Student’s T test. p<0.05 was considered statistically significant.

BMI, body mass index; HOMA-IR, homeostatic model assessment- insulin resistance; β2AR, beta-2 adrenergic receptor; NCM, non-classical monocytes (CD14^-CD16⁺⁺); IM, intermediate monocytes (CD14⁺CD16⁺); CM, classical monocytes (CD14⁺CD16^-); ILC2s, group 2 innate lymphoid cells; MFI, mean fluorescence intensity.

We identified and quantified the three populations of circulating monocytes to estimate systemic inflammation (Fig 2). Both non-classical monocytes (NCM) percentage and CD16⁺/CD16^- ratio were increased in obese subjects suggesting a deregulation between pro-inflammatory and anti-inflammatory subsets (Table 1).

Increased β2AR expression on intermediate monocytes

The β2AR expression on NCM (CD14^-CD16⁺⁺) and CM (CD14⁺⁺CD16^-) was not different within the groups of obese and non-obese subjects. Yet, in obese subjects, the IM (CD14⁺CD16⁺) expressed significantly more β2AR (p<0.001) than in non-obese subjects (Table 1 and Fig 3).

Fig 3 — PBMC were incubated for 1 h with anti-β2AR polyclonal Ab and next stained with a secondary F´(ab)2 PE mAb. Next, PBMC were labeled with anti-CD14-FITC and–CD16-PE mAbs. A) Representative dot plot depicts FSC and SSC to identify monocytes. B) PE-isotype control is shown in a histogram. C) Histograms correspond to β2AR staining within the each Mo subset as follows: the expression of β2AR on CD14^-CD16⁺⁺ or NCM (left panels), on CD14⁺CD16⁺ or IM (middle panels), and on CD14⁺⁺CD16^- or CM (right panels). MFI is indicated. Results shown are from a representative lean individual (top panels) and a subject with obesity (bottom panels). MFI = mean fluorescence intensity.

Quantification of circulating group 2 innate lymphoid cells (ILC2s)

We defined ILC2s subset as Lin^-CD127⁺CRTH2⁺IL-5⁺ cells (Fig 4A). Flow cytometric analysis revealed the presence of ILC2s in peripheral blood, which are significantly diminished in obese subjects in comparison with lean subjects (p<0.0001) (Table 1 and Fig 4B).

Relationship between monocytes subsets, ILC2s percentages and β2AR expression with anthropometric and metabolic characteristics at baseline

We examined the association of ILC2s, monocyte subsets, and β2AR expression on Mo, with anthropometric and metabolic factors in the whole group of study, using a multiple regression model (Table 2).

Table 2. Relationships of monocytes subsets with anthropometric, and metabolic features in basal state.

Dependent variable	Regressors	β±S.E.	T	p-level
*Non-classical monocytes* (NCM; CD14^-CD16⁺⁺) Adjusted R² = 0.135
	Intercept		4.97	<0.000002
	HOMA-IR	0.33±0.08	4.26	<0.000037
	Acylated-ghrelin	-0.16±0.08	1.98	<0.04
*Intermediate monocytes* (IM; CD14⁺CD16⁺) Adjusted R² = 0.127
	Intercept		2.62	<0.0098
	Caloric intake	0.17±0.08	2.00	<0.047
*CD16⁺/CD16^- ratio* Adjusted R² = 0.080
	Intercept		0.27	0.78
	BMI	0.35±0.09	3.73	<0.00028
	Leptin	-0.19±0.09	-2.06	<0.041
*ILC2s* Adjusted R² = 0.098
	Intercept		4.06	<0.000082
	Caloric intake	-0.24±0.08	-2.87	<0.0046
	HDL-C	0.16±0.08	1.97	<0.05
*β2AR expression on NCM* Adjusted R² = 0.119
	Intercept		-2.37	<0.019
	Mean arterial tension	0.28±0.08	3.35	<0.001
	Leptin	0.22±0.08	2.54	<0.012
	HDL-C	0.21±0.08	2.50	<0.013
	HOMA-IR	-0.18±0.09	-2.01	<0.045
*β2AR expression on IM* Adjusted R² = 0.322
	Intercept		-0.70	0.48
	HDL-C	0.42±0.08	5.13	<0.000001
	BMI	0.41±0.09	4.52	<0.00001
	REE	-0.25±0.08	-2.92	<0.004
*β2AR expression on CM* Adjusted R² = 0.360
	Intercept		-1.38	<0.016
	HDL-C	0.30±0.08	3.45	<0.0007
	BMI	0.24±0.09	2.63	<0.009
	Age	0.21±0.08	2.45	<0.015
	Non-HDL-C	-0.17±0.08	-2.08	<0.039

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The associations were evaluated by multiple regression. p<0.05 was considered statistically significant.

BMI, body mass index; EE, energy expenditure; β2AR, beta-2 adrenergic receptor; NCM, non-classical monocytes (CD14^-CD16⁺⁺); IM, intermediate monocytes (CD14⁺CD16⁺); CM, classical monocytes (CD14⁺CD16⁺); ILC2s, group 2 innate lymphoid cells.

Non-classical monocytes (CD14^-CD16⁺⁺) correlated positively with HOMA-IR, but negatively with acylated-ghrelin levels. Intermediate monocytes (CD14⁺CD16⁺) were related to caloric intake. CD16⁺/CD16^- ratio correlated positively with BMI, and inversely with leptin levels. ILC2s percentage was associated positively with HDL-C levels but inversely with caloric intake.

On the other hand, β2AR expression on NCM was associated with HDL-C, leptin levels, and mean arterial tension. There were positive relationships between β2AR expression on IM with HDL-C, and BMI, but negatively with REE. β2AR expression on CM was associated with HDL-C levels, BMI, and age.

After testing for confounding factors, the models did not change.

Effects of hypocaloric diet over monocyte subsets, circulating ILC2s and β2AR expression

To determine the effect of the hypocaloric diet on the monocyte subsets, ILC2s, and β2AR expression, we compared values before and 7-weeks after diet. Thirteen individuals with obesity were excluded for unsatisfactory adherence to diet, resulting in a sample size of 52 subjects. At the end of intervention, subjects showed a mean weight loss of 4.0 kg, corresponding to a 4.5%. REE did not change, but triglycerides and insulin resistance decreased significantly (Table 3).

Table 3. Changes in anthropometric, biochemical, and hormonal measures, monocyte subsets, ILC2 and β2AR expression after caloric restriction.

Variable	Before CR Mean±S.D.	After CR Mean±S.D.	Δ	t	p-value
Weight (kg)	90.1±14.5	86.0±14.8	-4.05	16.7	<0.0001
BMI (kg/m²)	33.9±4.7	32.3±4.7	-1.5	17.8	<0.0000000001
Mean arterial tension (mmHg)	90.1±6.4	86.2±5.4	-3.8	3.9	<0.00022
REE (kcal/day)	1768±377	1722±329	-46	1.24	0.22
Glucose (mg/dl)	90±13	88±13	-2.4	1.2	0.23
HDL-cholesterol (mg/dl)	51±12	46±13	-12	3.1	<0.0033
Non-HDL-cholesterol (mg/dl)	142±41	134±29	-7.8	1.8	0.07
Triglycerides (mg/dl)	186±218	138±83	-48	2.1	<0.039
Insulin (μUI/ml)	15.2±9.1	13.1±9.0	-2.1	2.6	<0.011
HOMA-IR	3.5±2.2	2.9±2.1	-0.6	3.2	<0.0024
Ghrelin (μg/ml)	84.8±43.3	81.7±45.8	-3.1	0.9	0.38
Leptin (pg/ml)	43.5±24.9	41.1±24.2	-2.4	0.9	0.35
Group 2 Innate lymphoid cells (ILC2) (%)	2.7±2.5	5.8±4.5	2.8	-2.9	<0.008
Non-classical monocytes (CD14^-CD16⁺⁺) (%)	7.2±6.1	5.3±3.7	-1.8	2.4	<0.02
Intermediate monocytes (CD14⁺CD16⁺) (%)	8.0±4.7	7.5±5.5	-0.5	0.5	0.61
Classical Monocytes (CD14⁺CD16^-) (%)	62.9±15.3	71.0±10.6	6.7	-3.3	<0.0015
CD16⁺/CD16^- ratio	0.3±0.2	0.2±0.1	-0.08	2.5	<0.015
β2AR expression on NCM (MFI)	10778±5489	10501±6180	-1743	0.25	0.80
β2AR expression on IM (MFI)	27069±15070	23350±13916	-6480	1.7	<0.05
β2AR expression on CM (MFI)	14343±8117	13626±8496	-2785	0.5	0.60

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n = 52 obese subjects. Differences between groups were evaluated with Student’s T test. p<0.05 was considered statistically significant. REE, resting energy expenditure

CR, caloric restriction; BMI, body mass index; HOMA-IR, homeostatic model assessment- insulin resistance; EE, energy expenditure; β2AR, beta-2 adrenergic receptor; NCM, non-classical monocytes (CD14^-CD16⁺⁺); IM, intermediate monocytes (CD14⁺CD16⁺); CM, classical monocytes (CD14⁺CD16⁺); MFI, mean fluorescence intensity.

We also evaluated possible modification of the abundance of Mo subsets and ILC2s and β2AR expression on monocytes after diet (Table 3 and Fig 5). We found that NCM (CD14^-CD16⁺⁺) decreased and CM (CD14⁺CD16^-) increased significantly (Fig 5A and 5B). Yet, IM had a non-significant decrease (Fig 5C). There was also a significant diminution in the CD16⁺/CD16^- ratio (Fig 5D). Furthermore, circulating ILC2s increased (Fig 5F), and β2AR expression on IM significantly decreased (Fig 5E).

Associations of changes in monocyte subsets, ILC2s percentage and β2AR expression after dietary intervention

Table 4 shows the changes in NCM (CD14^-CD16⁺⁺), IM (CD14⁺CD16⁺), and CD16⁺/CD16^- ratio after diet, negatively associated with ΔHDL-C and Δleptin levels. The change in CM (CD14⁺CD16^-) was positively associated with ΔHDL-C concentrations. Interestingly CD16⁺/CD16^- ratio showed associations positive with ΔREE but negative with ΔBMI. The ΔCM (CD14⁺CD16^-) was positively associated only with ΔHDL-C. The changes in ILC2s were negatively correlated with ΔHOMA-IR. The changes in the β2AR expression on NCM and CM were associated negatively with ΔBMI and positively with Δleptin in both cases. Also the Δβ2AR expression on IM was associated negatively with ΔBMI. The Δβ2AR expression on NCM was positively related with ΔHDL-C, but negatively with ΔHOMA-IR.

Table 4. Associations of changes in monocytes subsets, ILC2s, and β2AR expression within Mo subsets with anthropometric and metabolic features after dietary restriction.

Dependent variable	Regressors	β±S.E.	T	p-level
*ΔNon-Classical monocytes* (NCM; CD14^-CD16⁺⁺). Adjusted R² = 0.94
	Intercept		-4.07	<0.00013
	ΔLeptin levels	-0.27±0.12	-2.22	<0.03
	ΔHDL-C	-0.24±0.12	-1.96	<0.046
*ΔIntermediate monocytes* (IM; CD14⁺CD16⁺) Adjusted R² = 0.134
	Intercept		2.25	<0.04
	ΔLeptin levels	-0.34±0.12	-2.91	<0.01
	ΔHDL-C	-0,24±0.12	-2.02	<0.047
*ΔClassical monocytes* (CM; CD14⁺CD16^-) Adjusted R² = 0.157
	Intercept		3.79	<0.0001
	ΔHDL-cholesterol	0.37±0.11	3.20	<0.002
*ΔCD16⁺/CD16^- ratio* Adjusted R² = 0.316
	Intercept		-4.49	<0.00003
	ΔHDL-Cholesterol	-0.34±0.10	-3.29	<0.0017
	ΔLeptin	-0.31±0.11	-2.82	<0.0065
	ΔBMI	-0.32±0.11	-2.82	<0.0065
	ΔREE	0.23±0.11	2.08	<0.04
Δ*Group 2 innate lymphoid cells (ILC2s)* Adjusted R² = 0.143
	Intercept		7.37	<0.0000001
	ΔHOMA-IR	-0.40±0.12	-3.42	<0.0011
Δ*β2AR expression on NCM* Adjusted R² = 0.401
	Intercept		-5.04	<0.000005
	ΔBMI	-0.53±0.10	-5.12	<0.000003
	ΔLeptin	0.49±0.11	4.66	<0.000018
	ΔHDL-C	0.25±0.10	2.63	<0.011
	ΔHOMA-IR	-0.24±0.10	-2.37	<0.021
Δ*β2AR expression on IM* Adjusted R² = 0.058
	Intercept		-3.09	<0.0030
	ΔBMI	-0.27±0.12	-2.22	<0.029
Δ*β2AR expression on CM* Adjusted R² = 0.258
	Intercept		-4.54	<0.000026
	ΔBMI	-0.53±0.11	-4.65	<0.000018
	ΔLeptin	0.25±0.11	2.19	<0.032

Open in a new tab

The associations were evaluate by multiple regression (n = 52). p<0.05 was considered statistically significant.

Δ, delta; BMI, body mass index; REE, energy expenditure; β2AR, beta-2 adrenergic receptor; NCM, non-classical monocytes (CD14^-CD16⁺⁺); IM, intermediate monocytes (CD14⁺CD16⁺); CM, classical monocytes (CD14⁺CD16⁺).

Discussion

In this work we studied the three types of circulating monocytes and ILC2s, and the elevation of β2AR expression on intermediate monocytes as estimators of chronic low-grade inflammation in individuals with obesity, We analysed its associations with energy expenditure, anthropometry, hormonal and metabolic variables before and after of dietary restriction. The importance of chronic low-grade inflammation in obesity is well described, implicating perturbations of immune system and dysregulated adipose tissue (AT) homeostasis [30]. Our findings, using a comprehensive multiparameter cytometry analysis of Mo and ILC2s in peripheral blood, provide further support to their role in metabolic homeostasis and the physiopathology of AT.

We found circulating NCMs increased in the obese group, but CMs and IMs did not change. Previous studies on the abundance of circulating CM (CD14⁺CD16^-) in obesity are inconsistent, with reports showing decrease or no change [15]. Ours results, support the concept of increased circulating CD16⁺ monocyte subpopulations (IM and NCM) in obesity associated with cardio-metabolic risk factors [15–20]. The intermediate subtype (CD14⁺CD16⁺) is a small percentage of transitional monocytes with phagocytic and more pro-inflammatory capacity than the NCM [15,31,32]. Zawada et al [33] proposed a pro-angiogenic behaviour for IM, suggesting that the increase in peripheral blood has an important role for inflammation and progression into atherosclerosis [32]. In contrast to Krinninger et al [32], who found increase the percentage of CD14⁺CD16⁺ monocytes, we only found that IM count had a non-significant trend to increase. Despite these results, it is possible that in obesity occurs a shift of monocytes toward a pro-inflammatory phenotype.

Innate lymphoid type 2 cells (ILC2s) are key regulators of the immune and metabolic homeostasis of visceral adipose tissue (VAT) and may be determinants of weight, considering their involvement in beige fat development, through IL-5 [34–37]. Altered ILC2s amounts and function have been found in VAT but not in circulation in humans and other species with metabolic disorders such as obesity. In the present work, we developed an assay to assess circulating ILC2 by four-color flow cytometry following directions of previous scientific works in allergy [38–40]. We demonstrated that obese subjects have decreased circulating ILC2s. To our knowledge, this is the first report using this procedure; therefore we cannot compare our results with other investigations. Multiparameter flow cytometry of freshly drawn peripheral blood offers the promise of a highly sensitive and reproducible approach, which allows the identification and quantification of complex cell subpopulations, such as ILCs.

Obesity is also linked to altered hypothalamic–pituitary–adrenal axis (HPA) and sympathetic nervous system (SNS) function, triggering inflammation which increases β2AR expression in peripheral blood mononuclear cells [41]. In our study, intermediate monocytes (CD14⁺CD16⁺) from obese individuals expressed more β2AR. Our results agree with those of Gálvez et al [42] who described β2AR induction of a shift towards an anti-inflammatory phenotype profile. In addition, Hong et al [43] demonstrated higher amounts of β2AR on monocytes with reduced responsiveness. Due to the role of sympathetic activation in hypertension and cardiovascular pathology is well studied, and the IM are involved in cardiovascular events, the overexpression of β2AR is in agreement with the increased frequency of hypertension and cardiovascular damage in obese patients. Therefore, sympatho-adrenal regulation in monocytes is an important aspect of vascular inflammation. In addition, β2AR may act as a molecular rheostat to fine-tune anti-inflammatory responses preventing inflammation [44].

Inflammation modifies energy metabolism, enhancing energy expenditure, and reducing energy intake, and induces AT remodelling [2]. The associations of caloric intake, positive with IM subset, and negative with ILC2s, support the pro-inflammatory environment of the metabolic imbalance and loss of AT browning observed in obesity.

Ghrelin and leptin are important components of the neuroendocrine control of energy homeostasis and immune system regulation [9,10,45,46]. In our work, acylated-ghrelin was associated negatively with NCM, which we explain by the ghrelin action as an anti-inflammatory cytokine in homeostasis with potent orexigenic effect. Total ghrelin levels are reduced in obese patients. In addition, a lack of ghrelin signalling or increase conversion to the desacyl form may exacerbate the inflammatory response [10,46]. In contrast, leptin, a pro-inflammatory cytokine, has an important role in the control of energy metabolism and the metabolism-immune interplay [47]. Leptin promotes the proliferation and activation of NCM and IM favouring metabolic diseases, such as obesity [45,48], thereby leptin action induces higher BMI and REE as an effect of the chronic low-grade inflammation per se, which could explain the increase of β2AR expression on monocytes and its association with BMI.

We found a positive relationship between insulin-resistance and NCMs, suggesting that obesity-induced insulin resistance aggravates the chronic low-grade inflammation favoring the shift to increase the CD14^-CD16⁺⁺ subset (NCM) [21,49]. We also confirmed the relations of NCMs and ILC2s with plasma lipids. Increased CD14⁺CD16⁺⁺ monocytes and low HDL-C levels are reported in inflammatory disorders [22]. HDL-C and Apo A-1 may prevent monocytes activation and their attachment to the endothelium surface [50]. Furthermore, HDL-cholesterol levels are positively associated with ILC2s. This suggests that HDL-C are involved in cardiovascular protection due to its anti-inflammatory, antioxidant, and antithrombotic properties [35,51]. Furthermore, dietary intervention reverses HDL-C levels and ILC2s.

Since dietary habits influence energy and immune homeostasis, weight loss in obesity has a beneficial effect in the immune system, mediated by secretion of hormones and cytokines [52]. In this work, the inflammatory status decreased under caloric restriction mediated probably by the increase of CM and ILC2s. Few studies explore the impact of different types of diets on weight loss, the immune response [53] and immune cells [54,55]. We found that short-term caloric restriction induced a 4.5% weight loss diminishing CD14^-CD16⁺⁺ (NCM) and CD14⁺CD16⁺ (IM) monocyte numbers, pointing to a decrease of chronic low-grade inflammation. In contrast, Manco et al [56] reported that a 5% weight loss do not induce an effect on systemic or subcutaneous adipose tissue markers of inflammation. However, our findings agree with other works e.g. Nieman et al [57] mentioned that a moderate weight loss, decrease certain aspects of immune system (percentage of immune cells and functions); and Kim et al demonstrated that caloric restriction with a high protein diet, also decreases NCM subset [22]. Considering that we administered a diet with moderate caloric restriction we concluded that this diet might offer the benefit of reduction of inflammatory damage, even with moderate weight loss.

In our work, circulating ILC2s augmented after dietary intervention, which led a decrease of REE by the mediation of IL-5 secretion [26,51], therefore, ILC2s might balance visceral metabolic homeostasis and promote beige cells expansion, which regulates REE. The findings of Brestoff et al. [26] also support the idea that ILC2s may be a key immune component of the thermogenic circuit and a determinant of adipose tissue metabolic status.

The decreased β2AR expression on IM after caloric restriction may contribute to mitigate inflammation. We found that after caloric restriction the changes of β2AR expression on three subpopulations of monocytes correlated with diminution of BMI, in agreement with previous reports [42,57].

Conclusions

This study shows several novel findings: the increase of NCM, decreases of circulating ILC2s, and higher β2AR expression on IM in obese subjects, the positive associations between caloric intake and IM, and negative with ILC2s, gives further support to the concept of the participation of inflammation in energy expenditure. We observed after intervention a decrease of circulating NCM, and an increase of CM, and ILC2s. In conclusion, our work underscores the link between obesity and pro-inflammatory environmental that influences impairments of immune system and metabolism, responses that may be modified after dietary intervention.

Supporting information

S1 Table

(XLSX)

Click here for additional data file.^{(146.1KB, xlsx)}

Data Availability

All relevant data are available within the paper and its Supporting Information files.

Funding Statement

This work was supported by the grant CB2014-242065M from Consejo Nacional de Ciencia y Tecnología (CONACYT, México, to Juan M. Malacara). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0228637.r001

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24 Oct 2019

PONE-D-19-23742

Analysis of the percentages of monocyte subsets and ILC2s, their relationships with metabolic variables and response to hypocaloric restriction in obesity

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Additional Editor Comments:

In addition to the points made by the reviewers, please add more details for your flow cytometery methodology. How did you set your gates? For example, were FMOs only used for ICL2s? The gating strategy needs more detail, and explaining the methods will help strengthen the manuscript.

Why are all your cells in Figure 2 positive? It seems there should be cells that are not β2AR positive.

In addition, your monocyte gate is quite small, which is particularly worrisome since the voltage is such that there is not much “separation” between cell populations. (for future research studies, you may want to consider adjusting your voltages so that it is easier to distinguish between cell populations)

Please carefully go through your results and discussion sections and make the results more specific. As it is written right now, it is unclear which results are being compared. For example “No differences in β2AR expression by NCM (CD14-CD16++) and CM (CD14++CD16-) were observed between the groups of obese and non-obese subjects. Yet, the IM (CD14+CD16+) from obese subjects expressed significantly more β2AR (p<0.001) (Table 1 and Fig. 2).” These sections are missing which time points are being compared. This is a consistent problem throughout the results and discussion sections.

Much of the discussion session needs to be re-written. The current discussion section is written as a literature review, as there is very little discussion of the results of your study and your interpretation of what these findings mean. Although the discussion section generally avoids over-stating, it does need more work to discuss the findings of the research study.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I appreciate the opportunity to review this interesting paper on circulating monocytes and group 2 innate lymphoid cells (ILC2s) in lean and obese (obesity assessed by BMI) subjects, and the possible effect of hypocaloric restriction on these cells. The premise of this study is the hypothesis that in obese subjects non-classical monocytes subpopulation is increased and ILC2s are decreased comparing to lean controls and that the weight loss induced by hypocaloric restriction hypocaloric would reverse the situation. Authors has tested this hypothesis on 74 lean and 65 obese individuals, who were subject to diet restrictions for 7-weeks. They observed that In obese individuals the percent of non-classical monocytes (NCM), and the expression of the �2-adrenergic receptor (�2AR) expression by intermediate monocytes (IM) were increased, whereas the percent of ILC2s was decreased. Caloric restriction lead to a decrease of NCM and the �2AR expression by IM, as well as, an increase in the percent of classical monocytes and ILC2s. Authors concluded that the weight loss induced by hypocaloric restriction is significantly associated with changes of monocytes and ILC2s percentages, which may contribute to the attenuation of the low-grade inflammation linked to obesity.

The main strengths of this paper is that it addresses an interesting and timely problem, the article is well constructed, the experiments were well conducted, and analysis was well performed. Recently, ILC2s were identified in murine and human adipose tissue and have been shown to promote the white adipose tissue beiging and prevent the development of obesity

Considering these strengths, I feel some concerns on the manuscript, which should be addressed before being accepted for the publication.

Major comments:

• The research question is not clear from the introduction. Authors should more convincingly justify why they decided to measure subpopulations of monocytes and ILC2. Why do the authors think that it is important to measure circulating ILC2s in the blood when their action appears to be mainly local, in adipose tissue? Please discuss. Authors should also justify in the introduction why decide to measure leptin and ghrelin.

• Reliance on BMI as a sole marker of obesity, seems to be the serious limitation of the study. A number of authors indicated a poor linear relationship between BMI and total body fat and also suggested that body fat distribution would be more clinically significant than overall obesity. For instance using dual energy x-ray absorptiometry (DXA) seems to be fast and relatively inexpensive method to assess visceral adipose tissue.

• A flow chart showing the experimental procedure should be included as it makes the experimental setting more visible to the reader. It isn’t clear for me what were the effects of caloric restriction in non-obese participants?

• Conclusions are too general.

Minor comments:

• How exactly participants were selected for the study? I assume that individuals with BMI ≥24.9kg/m2 and ≤30 (kg/m2 were excluded from the study. Authors should clearly state it. In parasite infections and allergies, circulating ILC2s could be elevated. How did authors ensure exclusion such participants?

• Figures are confusing and the figure legends do not provide sufficient information to describe the data.

Reviewer #2: The purpose of the current study was to assess monocyte subsets and ILC2s in lean and obese subjects, and the possible

effect of energy restriction on these innate cells. The authors found that the percentage of non-classical monocytes and the expression of the Beta-2AR by intermediate monocytes were increased. In contrast the the percentage of ILC2s

was decreased in subjects with obesity. The authors also showed that there were significant negative associations between

ILC2 and caloric intake, and Beta-2AR expression by intermediate monocytes and resting energy expenditure, but a positive relationship between non-classical monocytes and insulin resistance. Caloric restriction reduced non-classical monocytes, and Beta-2AR expression by intermediate monocytes. Classical monocyte and ILC2 populations increased with caloric restriction as well. These results suggest that weight loss may contribute to the reduction in low-grade inflammation in part by increasing ILC2 cell numbers and modifying the inflammatory characteristics of circulating monocytes. Overall, these results are novel and interesting but there were numerous grammatical errors (too many to list) throughout the manuscript that should be corrected.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

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PLoS One. 2020 Feb 19;15(2):e0228637. doi: 10.1371/journal.pone.0228637.r002

Author response to Decision Letter 0

22 Nov 2019

Additional Editor Comments:

R = We included this information in the new version of manuscript, (M&M, results and figure legends sections) (page 6 lines 129-134, and 140-144; page 7 lines 145-148 and 156-161).

Why are all your cells in Figure 2 positive? It seems there should be cells that are not β2AR positive.

R = We used unstained cells tube and isotype control tube in each experiment for assigning the gates and markers for negative and positive fluorescence.

R = We re-edited the dot plots and gates with Kaluza software included the total events acquired (50,000). These figures were included in the new version of Figure 1 and figure 2.

R = We apologized by unclear redaction. We changed this paragraph in the new version of manuscript (page 9 lines 190-192).

R = According to this observation, we included in the new version of manuscript a discussion more detailed about our findings.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

R = According to this observation, we included in the new version of manuscript a discussion more detailed about our findings.

Reviewer #2: Yes

________________________________________

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

________________________________________

3. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

________________________________________

4. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: No

R = We reviewed the typographical and grammatical mistakes and we offer you a new corrected version.

________________________________________

5. Review Comments to the Author

Reviewer #1: I appreciate the opportunity to review this interesting paper on circulating monocytes and group 2 innate lymphoid cells (ILC2s) in lean and obese (obesity assessed by BMI) subjects, and the possible effect of hypocaloric restriction on these cells. The premise of this study is the hypothesis that in obese subjects non-classical monocytes subpopulation is increased and ILC2s are decreased comparing to lean controls and that the weight loss induced by hypocaloric restriction hypocaloric would reverse the situation. Authors has tested this hypothesis on 74 lean and 65 obese individuals, who were subject to diet restrictions for 7-weeks. They observed that In obese individuals the percent of non-classical monocytes (NCM), and the expression of the β2-adrenergic receptor (β2AR) expression by intermediate monocytes (IM) were increased, whereas the percent of ILC2s was decreased. Caloric restriction lead to a decrease of NCM and the β2AR expression by IM, as well as, an increase in the percent of classical monocytes and ILC2s. Authors concluded that the weight loss induced by hypocaloric restriction is significantly associated with changes of monocytes and ILC2s percentages, which may contribute to the attenuation of the low-grade inflammation linked to obesity.

Considering these strengths, I feel some concerns on the manuscript, which should be addressed before being accepted for the publication.

Major comments:

R = Both explanation were added in Introduction and Discussion sections (page 4, lines 78-80, and page 12 lines 239-242).

R = We agree with the reviewer contention that BMI has a poor linear relationship with total body mass. However, we do not agree with the affirmation that it is a serious limitation for the diagnosis of obesity. Current criteria widely used for the diagnosis of obesity, is based on BMI and not on the assessment of visceral body fat with DXA.

R = We included at final of Ms this flow chart which explains the recruitment of subjects and procedures. Diet was prescribed only for obese individuals as stated in M&M, Normal weight subjects continued with their customary diet.

• Conclusions are too general.

R = We offer you a new version of conclusions.

Minor comments:

R = We explained in more detail the selection in participants section and in the flow chart added at the end of M&M section. Subjects with overweight were not included in the study (≥24.9 kg/m2 and ≤30 kg/m2). Only recruitment individuals with >30BMI<35 kg/m2). In addition, as stated in M&M, we did not included subjects with infections (allergies, flu, cold, dental diseases, etc).

• Figures are confusing and the figure legends do not provide sufficient information to describe the data.

R = We re-edited the figures and M&M and figure legends sections in the new version of manuscript (page 6, lines 129-134 and 139-144; page 7, lines 145-149, and 150-161; page 32, lines 524-528, 532-535 and 539-548).

Reviewer #2.

The purpose of the current study was to assess monocyte subsets and ILC2s in lean and obese subjects, and the possible effect of energy restriction on these innate cells. The authors found that the percentage of non-classical monocytes and the expression of the Beta-2AR by intermediate monocytes were increased. In contrast the percentage of ILC2s was decreased in subjects with obesity. The authors also showed that there were significant negative associations between ILC2 and caloric intake, and Beta-2AR expression by intermediate monocytes and resting energy expenditure, but a positive relationship between non-classical monocytes and insulin resistance. Caloric restriction reduced non-classical monocytes, and Beta-2AR expression by intermediate monocytes. Classical monocyte and ILC2 populations increased with caloric restriction as well. These results suggest that weight loss may contribute to the reduction in low-grade inflammation in part by increasing ILC2 cell numbers and modifying the inflammatory characteristics of circulating monocytes. Overall, these results are novel and interesting but there were numerous grammatical errors (too many to list) throughout the manuscript that should be corrected.

R = We offer you a new corrected version of manuscript ________________________________________

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Attachment

Submitted filename: 19 11 Rebuttal_Mo_REE.docx

Click here for additional data file.^{(23.9KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0228637.r003

Decision Letter 1

Melissa M Markofski

26 Nov 2019

PONE-D-19-23742R1

Analysis of the percentages of monocyte subsets and ILC2s, their relationships with metabolic variables and response to hypocaloric restriction in obesity

PLOS ONE

Dear Dr Malacara,

I have received your revised manuscript, however there are a few things that need to be addressed before it is sent out for peer review. Figures are missing from the revised manuscript (only the flow chart is included in the revised manuscript, which is not given a figure number). In addition, in the authors' response it is stated that the "Increased β2AR expression by intermediate monocytes" paragraph has been revised. However, it is still exactly the same wording and still does not tell the reader what time points are different. Please go through the responses to make sure that all revisions that stated were completed were actually completed in the manuscript.

We would appreciate receiving your revised manuscript by Jan 10 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Melissa M Markofski

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

PLoS One. 2020 Feb 19;15(2):e0228637. doi: 10.1371/journal.pone.0228637.r004

Author response to Decision Letter 1

10 Jan 2020

January 10th, 2019

Additional Editor Comments:

In addition to the points made by the reviewers, please add more details for your flow cytometry methodology. How did you set your gates? For example, were FMOs only used for ICL2s? The gating strategy needs more detail, and explaining the methods will help strengthen the manuscript.

R = According to this observation, we have included this information in the corrected manuscript, in the Materials and Methods section (page 6, lines 130-135, and 139-143; page 7, lines 144-146 and 147-158), and figure legends (page 31, lines 513-516, 520-524, and 531-536).

Why are all your cells in Figure 2 positive? It seems there should be cells that are not β2AR positive.

R = According to this comment, we have added a new histogram of β2AR staining showing the percents of positive and negative cells, according to a negative (cells stained with an irrelevant isotype-matched mAb. In addition, and as stated in the corrected manuscript, results have been expressed as the MFI of positive cells. We used unstained cells tube and isotype controls tube in each experiment for assigning the gates and markers for negative and positive fluorescence. In the new Figure 2 we now included a histogram of PE-isotype control with its marker corresponding. Also in Figure Legend 2 included this information (page 31, lines 527-536).

R = We fully appreciate this recommendation. According to it, the dot plots and gates have been corrected, by using the Kaluza software. These figures have been included in the new version of the manuscript (Figs. 1 and 2).

R = We apologize by the confuse and improper writing of these paragraphs. According to this observation, these paragraphs have been corrected (page 8, lines 187-189).

R = According to this observation, the Discussion section has been thoroughly revised, with a proper analysis of our findings.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

R = According to this observation, we included in the new version of manuscript a discussion more detailed about our findings.

Reviewer #2: Yes

________________________________________

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

________________________________________

3. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

________________________________________

4. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: No

R = We have reviewed the grammar and syntax of the manuscript.

________________________________________

5. Review Comments to the Author

Reviewer #1: I appreciate the opportunity to review this interesting paper on circulating monocytes and group 2 innate lymphoid cells (ILC2s) in lean and obese (obesity assessed by BMI) subjects, and the possible effect of hypocaloric restriction on these cells. The premise of this study is the hypothesis that in obese subjects non-classical monocytes subpopulation is increased and ILC2s are decreased comparing to lean controls and that the weight loss induced by hypocaloric restriction hypocaloric would reverse the situation. Authors has tested this hypothesis on 74 lean and 65 obese individuals, who were subject to diet restrictions for 7-weeks. They observed that In obese individuals the percent of non-classical monocytes (NCM), and the expression of the β2-adrenergic receptor (β2AR) expression by intermediate monocytes (IM) were increased, whereas the percent of ILC2s was decreased. Caloric restriction lead to a decrease of NCM and the β2AR expression by IM, as well as, an increase in the percent of classical monocytes and ILC2s. Authors concluded that the weight loss induced by hypocaloric restriction is significantly associated with changes of monocytes and ILC2s percentages, which may contribute to the attenuation of the low-grade inflammation linked to obesity.

Considering these strengths, I feel some concerns on the manuscript, which should be addressed before being accepted for the publication.

Major comments:

R = According to this observation, we have added relevant information regarding adipokines (page 3, lines 51-58), and immune cells (page 4 lines 66-69, 78-80, and 86-88) in the Introduction as well as in the Discussion (page 11, lines 238-240, and 255-259; and page 12, lines 260-261). We hope that this reviewer detects now more convincing information in the corrected manuscript that justifies the analysis of the different immune and metabolic parameters detected in our study.

R = We agree that BMI has a poor linear relationship with total body fat. However, we consider that BMI is still a useful parameter for the diagnosis of obesity. In addition, unfortunately, in our University we do not have access to DXA.

R = According to this observation, a flow chart accounting for the recruitment of subjects and experimental procedures has been included in the corrected manuscript (page 33). Moreover, the caloric restriction diet was prescribed only for obese individuals, as stated in the Materials and Methods section of the corrected manuscript (page 5, lines 100-101, and 111-112, and page 6, lines 119-120).

• Conclusions are too general.

R = We are offering a new version of conclusions, with a proper analysis of our findings.

Minor comments:

R = According to this observation, a more detailed description of the inclusion criteria for the recruitment of individuals has been added to the corrected manuscript, including the new flow chart (page 33). Moreover, it is worth mentioning that subjects with overweight were not included in the study (≥24.9 kg/m2 and ≤30 kg/m2), and that those subjects with atopy, allergic symptoms or with parasite infections (according to their medical records) were also excluded from the study.

• Figures are confusing and the figure legends do not provide sufficient information to describe the data.

R = We re-edited the figures 1, 2 and 3, called now as New Fig. 1, New Fig. 2 and New Fig. 3. The information was added in M&M (page 6, lines 130-135, and 139-143; page 7, lines 146-147 and 150-159), and figure legends (page 312, lines 514-517, 520-525, and 528-537).

Reviewer #2.

R = We apologize for the numerous typos and grammar mistakes of the original manuscript. According to this observation, the manuscript has been thoroughly revised and we hope that this reviewer find a significant improvement in the grammar and syntax of the corrected manuscript.________________________________________

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Attachment

Submitted filename: 20 01 Rebuttal_Mo_REE.docx

Click here for additional data file.^{(27.6KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0228637.r005

Decision Letter 2

Melissa M Markofski

22 Jan 2020

Analysis of the percentages of monocyte subsets and ILC2s, their relationships with metabolic variables and response to hypocaloric restriction in obesity

PONE-D-19-23742R2

Dear Dr. Malacara,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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With kind regards,

Melissa M Markofski

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

**********

6. Review Comments to the Author

Reviewer #1: The authors have satisfactorily responded to all my questions and made the all necessary changes to the manuscript.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

PLoS One. doi: 10.1371/journal.pone.0228637.r006

Acceptance letter

Melissa M Markofski

30 Jan 2020

PONE-D-19-23742R2

Analysis of the percentages of monocyte subsets and ILC2s, their relationships with metabolic variables and response to hypocaloric restriction in obesity

Dear Dr. Malacara:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Melissa M Markofski

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table

(XLSX)

Click here for additional data file.^{(146.1KB, xlsx)}

Attachment

Submitted filename: 19 11 Rebuttal_Mo_REE.docx

Click here for additional data file.^{(23.9KB, docx)}

Attachment

Submitted filename: 20 01 Rebuttal_Mo_REE.docx

Click here for additional data file.^{(27.6KB, docx)}

Data Availability Statement

All relevant data are available within the paper and its Supporting Information files.

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PERMALINK

Analysis of the percentages of monocyte subsets and ILC2s, their relationships with metabolic variables and response to hypocaloric restriction in obesity

Nicté Figueroa-Vega

Carolina I Marín-Aragón

Itzel López-Aguilar

Lorena Ibarra-Reynoso

Elva Pérez-Luque

Juan Manuel Malacara

Roles

Abstract

Purpose

Methods

Results

Conclusions

Introduction

Materials and methods

Participants

Data collection

Fig 1. Flow chart for the clinical procedure.

Resting energy expenditure (REE)

Samples

Dietary intervention

Metabolic and hormonal measurements

Cell isolation

Identification of monocytes subsets

Evaluation of β2AR expression

Phenotypic characterization of ILC2s

Statistical analysis

Ethical approval

Results

Table 1. Comparison of basal characteristics among individuals.

Fig 2. Number of Mo subsets in lean and obesity state.

Increased β2AR expression on intermediate monocytes

Fig 3. β2AR expression among Mo subsets.

Quantification of circulating group 2 innate lymphoid cells (ILC2s)

Fig 4. Isolation and characterization of circulating ILC2s in peripheral blood.

Relationship between monocytes subsets, ILC2s percentages and β2AR expression with anthropometric and metabolic characteristics at baseline

Table 2. Relationships of monocytes subsets with anthropometric, and metabolic features in basal state.

Effects of hypocaloric diet over monocyte subsets, circulating ILC2s and β2AR expression

Table 3. Changes in anthropometric, biochemical, and hormonal measures, monocyte subsets, ILC2 and β2AR expression after caloric restriction.

Fig 5. Changes in Mo subsets, circulating ILC2s and β2AR expression after diet.

Associations of changes in monocyte subsets, ILC2s percentage and β2AR expression after dietary intervention

Table 4. Associations of changes in monocytes subsets, ILC2s, and β2AR expression within Mo subsets with anthropometric and metabolic features after dietary restriction.

Discussion

Conclusions

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Melissa M Markofski

Roles

Author response to Decision Letter 0

Decision Letter 1

Melissa M Markofski

Roles

Author response to Decision Letter 1

Decision Letter 2

Melissa M Markofski

Roles

Acceptance letter

Melissa M Markofski

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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