FIGURE LEGENDS
Fig 1. Circulating monocytes express metabolic
markers. Peripheral blood was obtained from healthy and dyslipidemic
individuals to analyse metabolic markers in circulating monocytes. (A)
Cells were analysed by multi-parametric flow cytometry. (B) Bloodstream
monocytes with metabolic phenotype (MoMe) levels according to ABCA1,
CD36 (n= 19) and PLIN2 (n= 20) expression in healthy subjects. (C) CD14
expression in metabolic monocytes. Data shown in panels B and C had a
non-Gaussian distribution and correspond to the median and Q1 and Q3.
Groups in panel C were compared using one-way ANOVA (Friedman test). *p
< 0.05, **p < 0.01, ***p < 0.001.
Fig 2. Metabolic monocytes in healthy individuals and in
patients with dyslipidemia. Bloodstream MoMe percentages from subjects
with elevated lipid levels (triglycerides >150 mg/dl, total
cholesterol >200 mg/dl) and healthy subjects. (A) MoMe
percentages evaluated with 2 (n= control 19, dyslipidemia 31) and 3 (n=
control 20, dyslipidemia 32) metabolic markers. Relative expression of
PPAR-γ mRNA in healthy individuals (n= 4) and in patients with
dyslipidemia (n= 8). (B) Different MoMe subsets according to their CD14
expression, divided into CD14 negative (n= control 20, dyslipidemia 31),
dim (n= control 19, dyslipidemia 31) and high (n= control 19,
dyslipidemia 31) expression. (C) Correlations between metabolic
monocytes percentages and total cholesterol (n= 29) or triglycerides (n=
29) or body mass index (D, n= 27) in subjects with dyslipidemia. All
data shown had a non-Gaussian distribution and correspond to the median
and Q1 and Q3. Groups were compared using Mann-Whitney test in panel A
and B. Spearman r analysis was performed in panel C. Linear regression
was performed. P value represents the likelihood of a nonzero slope.
Fig 3. Evaluation of M1- and M2-like monocytes levels in healthy
subjects and in patients with dyslipidemia. (A) Using flow cytometry,
starting from monocytes gate defined by FSC and SSC parameters, doble
cells were eliminated and then, CD14 expression was evaluated. (B) CD14
expression in M1-like monocytes (n=26) from dyslipidemia subjects and
CD14++CD68+CD80+comparation between patients (n= 26) and controls (n= 8). (C) CD14
expression in M2-like monocytes from subjects with dyslipidemia (n=26)
and
CD14+CD163+CD206+comparation between patients (n= 26) and controls (n= 8). Data shown in
bars graph B had a non-Gaussian distribution and correspond to the
median and Q1 and Q3. Data shown in individual values graphs and bars
graph C had a normal distribution and correspond to the arithmetic mean
and SD. Groups were compared using paired t test, Mann-Whitney
test, or unpaired t test. *p < 0.05, ***p <
0.001, ****p < 0.0001
Fig 4. Metabolic stimuli promote differentiation to
metabolically activated (MMe), classical (M1), and alternative
macrophages (M2). Monocytes were purified from healthy and dyslipidemia
subjects, cells were incubated with metabolic stimuli, dyed, and
analysed by flow cytometry for the expression of characteristic
polarization markers, M1
(CD14+CD68+CD80+),
M2
(CD14+CD163+CD206+)
and MMe
(CD14+ABCA1+CD36+PLIN2+).
(A) Cell culture conditions and MMe gating strategy. (B) Comparison of
MMe, M1 and M2 percentages, obtained from the incubation with metabolic
stimuli of monocytes from dyslipidemia patients (n= 9) and control
subjects (n= 6). Groups were compared using two-way repeated measures
ANOVA. *p < 0.05, ***p < 0.001
Fig 5. Metabolically activated (MMe), classical (M1),
and alternative (M2) macrophage differentiation in dyslipidemia and
healthy subjects. MMe (glucose, insulin, palmitate), M1 (IFN-γ, LPS)
and M2 (IL-4) macrophages obtained from culture from dyslipidemia and
healthy subjects. (A) MMe comparison between control and dyslipidemia
subjects evaluated with 2 (n= control 21, dyslipidemia 28; median and
IQR) and 3 (n= control 21, dyslipidemia 28; arithmetic mean and SD)
metabolic markers. (B) Correlation between MMe percentages and body mass
index (n= 24). (C) Correlation between MMe percentages and triglycerides
(n= 27), total cholesterol (n= 27), HDLc (n= 27) and LDLc (n= 26). (D)
ABCA1 median fluorescence intensity compared with HDLc (n= 27) and LDLc
(n= 25). (E) Monocyte-derived M1 (n= control 9, dyslipidemia 23) and M2
(n= control 9, dyslipidemia 22) macrophages in control and dyslipidemia
subjects. Groups were compared using Mann-Whitney test or unpairedt test. Spearman r analysis was performed. Linear regression was
performed. P value represents the likelihood of a nonzero slope.
*p < 0.05
Fig 6. Cytokine production by macrophages from
dislypidemic patients and healthy controls. Supernatants from
polarization cultures with metabolic stimuli or LPS were collected and
analysed by flow cytometry using a Cytometric Bead Array kit. (A) IL-12
quantification in M1 and MMe cell culture supernatants from healthy
subjects (n= 7). (B) IL-10 (n= 8) and IL-6 (n= 7) quantification in M1
and MMe cell culture supernatants from patients. Representative plot of
flow cytometry (n= 3) for
CD14+CD36+PLIN2+macrophages expressing IL-10 (red) compared with undyed control (blue).
(C) Cytokine concentrations produced by MMe from healthy and
dyslipidemia subjects. (D) IL-12 quantification in serum from control
(n= 7) and dyslipidemia subjects (n= 13). Data shown in A, B and D had a
non-Gaussian distribution and correspond to the median and Q1 and Q3.
Groups were compared using Wilcoxon matched-pairs signed rank test or
Mann-Whitney test. Groups were compared using two-way ANOVA. *p
< 0.05, ***p < 0.001