Download - Monocytes from Pregnant Women with Pre-Eclampsia are Polarized to a M1 Phenotype

Monocytes from Pregnant Women with Pre-Eclampsia arePolarized to a M1 PhenotypeLeonardo T. L. Medeiros1, Jos�e C. Perac�oli1, Camila F. Bannwart-Castro1, Mariana Rom~ao2, Ingrid C.Weel2, Marjorie A. Golim3, Leandro G. de Oliveira4, Cilmery S. Kurokawa1, Vera T. Medeiros Borges1,Maria T. S. Perac�oli2

1Department of Gynecology and Obstetrics, Botucatu Medical School, S~ao Paulo State University, Botucatu, Brazil;2Department of Microbiology and Immunology, Institute of Biosciences, S~ao Paulo State University, Botucatu, Brazil;3Division of Hemocenter, Botucatu Medical School, S~ao Paulo State University, Botucatu, Brazil;4Department of Gynecology and Obstetrics, Federal University of Sao Paulo, S~ao Paulo, Brazil

Keywords

Cytokines, monocyte subsets, pre-eclampsia,

surface receptors

Correspondence

Maria Terezinha S. Perac�oli, Department of

Microbiology and Immunology, Institute of

Biosciences, Rubi~ao Junior s/n, UNESP,

Botucatu, Sao Paulo, CEP 18618-970, Brazil.

E-mail: [email protected]

Submission November 22, 2013;

accepted February 3, 2014.

Citation

Medeiros LTL, Perac�oli JC, Bannwart-CastroCF, Rom~ao M, Weel IC, Golim MA, de Oliveira

LG, Kurokawa CS, Medeiros Borges VT,

Perac�oli MTS. Monocytes from pregnant

women with pre-eclampsia are polarized to a

M1 phenotype. Am J Reprod Immunol 2014;

72: 5–13

doi:10.1111/aji.12222

Problem

This study evaluated whether the monocyte inflammatory state in pre-

eclampsia (PE) might be associated with polarization to either M1 classi-

cally or M2 alternatively activated monocyte subsets.

Method of Study

Eighty-five women with (PE) and 52 normotensive (NT) pregnant

women matched for gestational age were included. Expression of surface

receptors characteristic of M1, such as Toll-like receptor (TLR)2, TLR4,

and CD64, or M2, such as CD163 and CD206 monocyte subsets were

evaluated in peripheral blood monocytes by flow cytometry. Tumour

necrosis factor-alpha (TNF-a), interleukin-(IL)-12p40, IL-12p70, and IL-

10 were evaluated in the supernatant of monocyte cultures by ELISA.

Results

Expression of TLR4 and CD64 by monocytes from pre-eclamptic women

was significantly higher, while the expression of CD163 and CD206

expression was significantly lower compared with NT pregnant women.

Endogenous production of TNF-a, IL-12p40, and IL-12p70 by monocytes

was increased, while synthesis of IL-10 was lower in women with PE

than in NT pregnant women.

Conclusions

Monocytes from women with PE are classically activated, producing

higher levels of pro-inflammatory cytokines, and express surface recep-

tors characteristic of the M1 subset. These results provide evidence that

the systemic inflammatory environment in PE may differentiate and

polarize these cells to the M1 phenotype.

Introduction

Pre-eclampsia (PE) is a specific syndrome of human

pregnancy affecting between 5 and 7% of primigr-

avid women1,2 and is characterized by new-onset

hypertension and proteinuria after 20 weeks of ges-

tation.3,4

Clinically, PE is classified as mild or severe,

according to patients’ signs and symptoms,4 and

more recently into early- or late-onset PE, depend-

ing on the time of appearance of clinical manifesta-

tions: early-onset PE tends to develop before

34 weeks of gestation, and late-onset PE develops

from 34 weeks of gestation.5,6 According to Hup-

American Journal of Reproductive Immunology 72 (2014) 5–13

ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd 5

ORIGINAL ARTICLE

pertz,6 early- and late-onset PE are two entities that

differ in aetiology and disease manifestation. Early-

onset PE is considered a foetal disorder that is typi-

cally associated with placental dysfunction, abnormal

uterine and umbilical artery Doppler evaluation, and

foetuses with growth restriction, intrauterine foetal,

or maternal complications.7,8 On the other hand,

late-onset PE is considered a maternal disorder more

frequently associated with a normal placenta, nor-

mal uterine and umbilical artery Doppler evaluation,

a low rate of foetal compromise, and more favour-

able perinatal outcome.1,8,9

Association between increased levels of serum

pro-inflammatory cytokines and chemokines as well

as enhanced leucocyte activation and oxidative stress

has been reported in PE.10–14 This systemic inflam-

matory reaction is similar to that observed in normal

pregnancy, but shows greater intensity.15,16 There-

fore, this disease might be resulting from an exacer-

bated maternal inflammatory response that includes

activation of inflammatory cells such as monocytes

and granulocytes, and endothelial cells.17,18 Thus, an

abnormal production of cytokines as well as imbal-

ance between pro-inflammatory cytokines, such as

tumour necrosis factor-alpha (TNF-a), interleukin-

(IL)-12, interferon-gamma (IFN-c), and IL-2 as well

as anti-inflammatory cytokines (IL-4, IL-10, and IL-

13),is involved in the vascular damage observed in

PE.19

The excessive activation of intravascular mono-

cytes and granulocytes as well as macrophages in PE

suggests that innate immune system activation can

cause problems in pregnancy progression. However,

the significance of these immunological changes in

the pathophysiology of PE is still unknown. In previ-

ous studies, we demonstrated that monocytes from

pregnant women with PE were endogenously acti-

vated and released high levels of reactive oxygen

intermediates associated with high production of

TNF-a and might be the major source of this cyto-

kine detected in patients’ plasma.13,20,21

Monocytes and macrophages are cells belonging to

the monocytic–macrophage lineage and are consid-

ered cell populations that may be adapted and

respond to a wide variety of signs in their environ-

ment.22 Macrophages can be classified into at least

two major subpopulations with distinct phenotypes

and functions, that is, classically activated/inflamma-

tory (M1) and alternatively activated/regenerative

(M2).22–25 This M1 and M2 classification refers to

the two extremes of a macrophage activation spec-

trum. These polarized cells differ by surface receptor

expression, cytokines and chemokines production, as

well as transcription and epigenetic pathways.25 M1

macrophages, activated by IFN-c, TNF-a, or lipopoly-saccharide (LPS), express opsonin Fcc-type receptors

RI (CD64), RII (CD32), and RIII (CD16), toll-like

receptor 2 (TLR2) and toll-like receptor 4 (TLR4),

inflammatory cytokines such as TNF-a, IL-6, IL-12,

and IL-23, as well as reactive oxygen and nitrogen

intermediates.22,26 On the other hand, activation by

IL-4 and IL-13 polarizes to the M2 profile, character-

ized by increased expression of CD163 scavenger

receptor for haemoglobin,27 which also has anti-

inflammatory and immunoregulatory functions,28

mannose receptor (CD206), as well as increased pro-

duction of IL-10 and transforming growth factor beta

(TGF-b1).22 This macrophage plasticity allows the

cell change from an activated state or M1 to an M2

regulatory state and vice versa, upon the influence

of specific environmental signals.29,30

The M1 and M2 classification, initially proposed

for macrophages, can be extended to human periph-

eral blood monocytes in diseases such as sepsis,31

infections,32 type 2 diabetes,33 and studies employ-

ing modulatory agents on the inflammatory response

to in vitro treatment of these cells.34,35 Considering

that peripheral blood monocytes are activated in PE

and produce higher levels of inflammatory cyto-

kines,13,20,21 in this study, we evaluated the two M1

and M2 monocyte phenotypes as well as their cyto-

kine profile in pre-eclamptic women.

Materials and methods

Patients and Controls

The study comprised 137 pregnant women without

previous history of hypertension or obstetric and

medical complications, admitted to the Obstetric Unit

of Botucatu Medical School, Botucatu, SP, Brazil

between March 2009 and October 2011. Eight-five

women were diagnosed with PE, defined as a persis-

tent increased blood pressure value of 140 9

90 mm Hg and proteinuria (≥300 mg in urine col-

lected during 24 hr) after the 20th week of gesta-

tion.36 Pre-eclamptic pregnant women were

classified according to the onset of clinical manifesta-

tions into early-onset (24–33 weeks of gestation,

n = 26) and late-onset (34–40 weeks of gestation,

n = 59) pre-eclampsia, according to the criteria sug-

gested by von Dadelszen et al.5 A group of 52 preg-


6 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

MEDEIROS ET AL.

nant women (24–33 weeks of gestation, n = 26),

and (34–40 weeks of gestation, n = 26) with an

uncomplicated pregnancy that remained normoten-

sive and non-proteinuric throughout pregnancy,

were recruited as controls and matched for gesta-

tional age with the pre-eclamptic group. Gestational

age was calculated from the last menstrual period

and confirmed by ultrasound dating. Blood samples

were collected from pre-eclamptic women when

diagnosed with early- or late-onset PE and from nor-

motensive women when they were paired with pre-

eclamptic women according to gestational age. Data

on the participating women are shown in Table I.

Proteinuria in 24-hr urine was measured by a calori-

metric method, the Technicon RAXT automation

system, and uric acid was assessed by uric acid enzy-

matic Trinder (Biotrol Diagnostic, Bridgewater, NJ,

USA) in the Clinical Laboratory, Hospital das Clini-

cas, Faculdade de Medicina de Botucatu – UNESP.

Exclusion criteria included multiple gestation, prior

PE, pregnant women in labour, illicit drug use, and

pre-existing medical conditions such as chronic

hypertension, diabetes, infectious and renal diseases.

The study was approved by the Ethics Committee of

the Botucatu Medical School, and informed consent

was obtained from all women involved in the study.

Isolation of Peripheral Blood Mononuclear Cells

Peripheral blood mononuclear cells (PBMC) were

isolated from heparinized venous blood by density

gradient centrifugation on Histopaque [density

(d) = 1.077] (Sigma–Aldrich, Chemical Co., St Louis,

Missouri, USA). Briefly, 10 mL of heparinized blood

was mixed with an equal volume of RPMI 1640 tis-

sue culture medium (Gibco Laboratories, Grand

Island, NY, USA) containing 2 mM L-glutamine,

10% heat-inactivated foetal calf serum, 20 mM 4-(2-

Hydroxyethyl)piperazine-1-ethanesulfonic acid (HE-

PES-Sigma-Aldrich), and 40 lg/mL gentamicin

(complete medium). Samples were layered over

5 mL Histopaque in a 15-mL conical plastic centri-

fuge tube. After centrifuging at 400 g for 30 min at

room temperature, the interface layer of PBMCs was

carefully aspirated and washed twice with phos-

phate-buffered saline (PBS) containing 0.05 mM

ethylenediaminetetraacetic acid (PBS-EDTA) and

once with complete medium, with centrifugation in

between washes at 300 g for 10 min. Cell viability,

as determined by 0.2% trypan blue dye exclusion,

was >95% in all experiments. Monocytes were

counted using neutral red (0.02%) in the PBMC sus-

pension, and a suspension of 5 9 105 monocytes/mL

in complete medium was employed for cytokine

determination and flow cytometry studies.

Monocyte Culture Supernatants

Monocyte suspensions (5 9 105/mL) were distrib-

uted (1 mL/well) in 24-well flat-bottomed plates

(Nalge Nunc, Rochester, NY, USA). After incubation

for 2 hr at 37°C in a humidified 5% CO2 atmo-

sphere, non-adherent cells were removed by aspira-

tion, and each well was rinsed twice with complete

medium. Monocyte preparations routinely contained

>90% monocytes, as determined by morphological

examination and staining for non-specific esterase.37

Monocytes were incubated with complete medium,

Table I Characteristics of Normotensive and Pre-eclamptic Pregnant Women

Groups

Normotensive Pre-eclamptic

Variable <34 weeks n = 26 ≥34 weeks n = 26 <34 weeks n = 26 ≥34 weeks n = 59

Age (years) 22 (17–40) 23 (15–40) 23 (14–43) 25 (15–43)

Gestational age (weeks) 29 (24–33) 37 (34–40) 31 (24–33) 37 (34–42)

Systolic blood pressure (mmHg) 105 (95–110) 100 (95–110) 160* (140–210) 150* (140–210)

Diastolic blood pressure (mmHg) 65 (60–70) 60 (60–70) 110* (90–140) 100* (90–130)

Proteinuria (mg/24 hr) <300 <300 1550* ** (320–20,160) 720* (300–17,550)

Uric acid (mg/dL) 3.2 (1.6–4.1) 3.4 (1.8–3.9) 5.9* (3.6–10.2) 5.0* (2.4–8.2)

Results are expressed as median (range).

*P < 0.05 compared with both groups of normotensive women (Kruskal–Wallis test).

**P < 0.05 compared with pre-eclamptic women with gestation age ≥34 weeks (Mann–Whitney U-test).



M1 MONOCYTE POLARIZATION IN PRE-ECLAMPSIA

without stimulus, for 18 hr at 37°C in 5% CO2. Cul-

ture supernatants were harvested and stored at

�80°C until assayed for cytokine determination.

Cytokine Determination

Cytokine concentrations in monocyte culture super-

natants were determined by enzyme-linked immu-

nosorbent assay (ELISA), using Quantikine ELISA

kits (R&D Systems, Minneapolis, MN, USA) for TNF-

a and IL-10, and ELISA MAX kits (Biolegend, Inc,

San Diego, CA, USA) for IL-12p40 and IL-12p70

according to the manufacturer’s instructions. Assay

sensitivity limits were 1.6 pg/mL for TNF-a, 3.9 pg/

mL for IL-10, 6.8 pg/mL for IL-p40, and 2 pg/mL for

IL-12p70.

Flow Cytometric Analysis of TLR2, TLR4, CD64,

CD163, and CD206 on Monocyte Surface

Monocyte surface expression of TLR2, TLR4, CD64,

CD163, and CD206 was assessed by flow cytometry

using a FACScalibur flow cytometer with Cell Quest

software (Becton Dickinson, Franklin Lakes, NJ,

USA). PBMCs containing 5 9 105 monocytes/mL

from normotensive and pre-eclamptic women were

distributed into non-adherent polystyrene tubes

(Becton Dickinson-ref 352054) and incubated for

18 hr at 37°C in a humidified 5% CO2 atmosphere

with complete medium. Cells were washed and

incubated with the following monoclonal antibodies

according to the manufacturer’s instructions: R-phy-

coerythrin/CyanineTM7 (PECy7)-labelled anti-CD14

(Biolegend), R-phycoerythrin (PE)-labelled anti-

TLR2 (Biolegend), fluorescein isothiocyanate (FITC)-

labelled anti-TLR4 (Biolegend), FITC-labelled anti-

CD64, PE-labelled anti-CD163 (Biolegend), or FITC-

labelled anti-CD206 (Biolegend). The cells were

incubated for 30 min in the dark at room tempera-

ture, washed, and then fixed with 2% paraformalde-

hyde in PBS. Background staining was determined

by staining cells for 30 min with FITC, PE, or

PECy7-labelled control isotype antibodies at room

temperature in the dark. All samples were then

washed twice with wash buffer containing 10%

endotoxin-free foetal bovine serum (Sigma-Aldrich),

fixed with saline buffer plus 2% paraformaldehyde

in PBS (Sigma-Aldrich) at room temperature for

20 min, and analysed by flow cytometry. Ten thou-

sand monocyte events, defined as cells with respec-

tive side scatter (SSC) and CD14 staining

characteristics, were acquired, and corresponding

levels of TLR2, TLR4, CD64, CD163, and CD206

were obtained from the CD14 cell gate. The results

are expressed as median fluorescence intensity (MFI)

in CD14+ cells analysed.

Statistical Analysis

Comparisons between characteristics of the PE and

NT groups, such as maternal age, gestational age,

serum uric acid, cytokine production by monocytes,

as well as monocyte surface markers, were analysed

by the nonparametric Kruskal–Wallis test. Protein-

uria concentration in early- and late-onset PE was

compared by the nonparametric Mann–Whitney U-

test. All analyses were carried out using INSTAT

3.05 software (GraphPad, San Diego, CA, USA). Sta-

tistical significance was accepted at P < 0.05.

Results

Clinical Characteristics of Pre-eclamptic and

Normotensive Pregnant Women

Characteristics of pre-eclamptic and NT pregnant

women showed no significant differences in relation

to age and maternal age. The concentration of pro-

teinuria was significantly higher in early-onset PE

than in late-onset PE. Serum uric acid levels were

similar in both groups of PE women and showed sig-

nificant differences compared with both groups of

NT pregnant women (Table I).

Production of Pro-Inflammatory Cytokines is

Augmented in Pre-Eclamptic Pregnant Women

Endogenous production of pro-inflammatory cyto-

kines TNF-a, IL-12p40, and IL-12p70 was signifi-

cantly higher while production of the anti-

inflammatory cytokine IL-10 by monocytes from

pre-eclamptic women was lower than in NT preg-

nant women (Fig. 1). Similar results were observed

when the groups with PE were distributed according

to gestational age in early- and late-onset PE. Both

groups of pre-eclamptic women showed significantly

higher levels of TNF-a, IL-12p40, and IL-12p70 com-

pared with NT pregnant women classified by gesta-

tional age (Fig. 2a–c). On the other hand, IL-10

production was significantly lower in both groups of

early- and late-onset PE than in NT pregnant

women (Fig. 2d). There was no significant difference



MEDEIROS ET AL.

between the two groups of pregnant women with

PE, or between the two groups of NT pregnant

women with regard to this basal cytokine produc-

tion.

Expression of Surface Markers on Monocytes of

Pre-eclamptic Pregnant Women

Monocytes from pregnant women with PE expressed

TLR4 and CD64 surface markers with higher expres-

sion than monocytes from NT pregnant women. No

significant differences were observed between these

groups in relation to TLR2 expression. On the other

hand, the MFI of CD163 and CD206 was lower in

the PE group than in the NT group. A representative

histogram with MFI of TLR2, TLR4, CD64, CD163,

and CD206, as well as the results of MFI expressed

by monocytes clearly, shows the differences between

pre-eclamptic and NT pregnant groups (Fig. 3).

When the pre-eclamptic group was classified as

early- and late-onset PE, similar results were

obtained. There were no statistically significant dif-

ferences in TLR2 expression between PE and NT

pregnant women with the same gestational age

(Fig. 4a). The expression of TLR4 and CD64 on the

monocyte surface, both in early- and late-onset PE,

was significantly higher than in the NT group with

equivalent gestational age. TLR4 was significantly

higher in early-onset PE compared with late-onset

PE. (Fig. 4b, c). Although there was a tendency to

higher expression of CD64 in the early-onset PE

group, the values were not significant different in

relation to the late-onset PE group.

The MFI of CD163 expression was significantly

lower in cells from pregnant women with early-

onset PE compared with NT women with gestational

age <34 weeks. No significant differences were

observed between late-onset PE and NT pregnant

women with the same gestational age (Fig. 4d).

Fig. 1 Cytokines produced by monocytes from patients with pre-

eclampsia (PE n = 85) and from normotensive (NT n = 52) pregnant

women cultured for 18 hr without stimulus. The results are expressed

as median (horizontal line), 25th and 75th percentile (box), and range

(whisker). *(P < 0.05) versus normotensive (Mann–Whitney U-test).

(a) (b)

(c) (d)

Fig. 2 Endogenous production of tumour necrosis factor-alpha (TNF-a) (a), interleukin (IL)-12p40 (b), IL-12p70 (c), and IL-10 (d) by monocytes from

patients with pre-eclampsia (PE) classified as early-onset (<34 weeks of gestation, n = 26) or late-onset (≥34 weeks of gestation, n = 59) pre-

eclampsia, and by monocytes from normotensive (NT) pregnant women (<34 weeks of gestation, n = 26) and (≥34 weeks of gestation, n = 26)

cultured for 18 hr without stimulus. The results are expressed as median (horizontal line), 25th and 75th percentile (box), and range (whisker). *

(P < 0.05) versus NT <34 weeks; +(P < 0.05) versus NT ≥34 weeks (Kruskal–Wallis test).




The CD206 receptor was significantly higher

expressed in NT pregnant women in the two gesta-

tional age periods evaluated compared with women

with PE evaluated in the same period. There was no

significant difference between the two groups of NT

pregnant women, nor between those with early- or

late-onset PE (Fig. 4e).

Discussion

Pre-eclampsia is one of the major causes of both

maternal and foetal morbidity and mortality.1 It is

clear that an exaggerated inflammatory response

occurs in this gestational pathology, but until now,

the probable causes remain unknown. Early- and

late-onset PE may differ in disease manifestation,

but so far it is not clear whether these two PE classi-

fications are two distinct disorders or simply a spec-

trum of disease severity across a range of gestational

ages.9

In the present study, urinary protein excretion at

the time of diagnosis was significantly higher in

cases of early-onset PE compared with late-onset PE.

These results confirm that proteinuria together with

blood pressure levels, in addition to being funda-

mental criteria for the diagnosis of PE, are consid-

ered important markers of disease severity.38 Serum

levels of uric acid were similar in both early- and

late-onset PE and were significantly higher than in

NT pregnant women. Hyperuricaemia is a common

finding in PE and is associated with oxidative stress,

TNF-a production, and disease severity.13

Peripheral blood monocytes from pregnant women

with PE are endogenously activated and secrete high

levels of free radicals and inflammatory cytokines,

suggesting their involvement in the disease patho-

physiology.13,14 In the present study, monocytes

from both groups of pregnant women with PE pro-

duced endogenously higher levels of TNF-a, IL-

12p40, and IL-12 p70 compared with the NT group.

Similar results were obtained by Giorgi et al.14 who

studied the production of TNF-a and IL-1b in associ-

ation with activation of nuclear transcription factor-

kappa B (NF-kB) in pregnant women with PE. The

authors showed that both endogenous production of

TNF-a and activation of NF-kB were significantly

(a)

(b)

Fig. 3 (a) Expression of toll-like receptor 2 (TLR2), toll-like receptor 4 (TLR4), CD64, CD163, and CD206 by monocytes from patients with pre-

eclampsia (PE n = 85) and from normotensive pregnant women (NT n = 52). The results are expressed as median of fluorescence intensity (MFI)

(horizontal line), 25th and 75th percentile (box), and range (whisker). (b) Representative histogram profile of TLR2, TLR4, CD64, CD163, and CD206

expressed by monocytes from patients with pre-eclampsia and by monocytes from normotensive pregnant women. *(P < 0.05) versus

normotensive (Mann–Whitney U-test).



MEDEIROS ET AL.

higher in pre-eclamptic pregnant women. The higher

levels of IL-12p40 and IL-12p70 in pre-eclamptic

pregnant women are in line with the results of Sakai

et al.,39,40 who suggested that increased production of

IL-12 and IL-18 by peripheral blood mononuclear

cells of pre-eclamptic pregnant women contributed to

the dominance of a Th1 profile in this pathology. It

has been reported that the exacerbated inflammatory

response in PE is associated with the release of sub-

stances with inflammatory activity, such as fragments

of the syncytiotrophoblast membranes, foetal DNA,

and soluble microparticles derived from leucocytes

and cytokines, which systemically activate inflamma-

tory cells such as monocytes, granulocytes, and endo-

thelial cells.10,15,18 Increased levels of heat-shock

protein 70 (Hsp70) associated with higher levels of

TNF-a, IL-1b, IL-12, and soluble TNF receptor-I (sTN-

FRI) have been described in serum from women with

early-onset PE compared with those with late-onset

PE.41 The higher levels of Hsp70 may originate from

the ischaemic and oxidatively stressed pre-eclamptic

placenta42 and are released into the circulation in

women with PE by increased shedding of syncytio-

trophoblast membrane microparticles, leading to

peripheral blood cell activation. These results support

a pro-inflammatory role of circulating Hsp70 in PE.43

Expression of TLR4 and CD64 on the surface of

non-stimulated monocytes, both in early- and late-

onset PE, was significantly higher than in NT preg-

nant women with equivalent gestational age. The

expression of TLR4 was also significantly higher in

early-onset PE than in late-onset PE. The high

expression of CD64 and TLR4 on the monocyte sur-

face was associated with increased endogenous pro-

duction of pro-inflammatory cytokines TNF-a, IL-

12p40, and IL-12p70, showing M1 monocyte fea-

tures, in pre-eclamptic pregnant women. These

results confirm the activation state of monocytes in

Fig. 4 Expression of toll-like receptor (TLR)2 (a), TLR4 (b), CD64 (c), CD163 (d), and CD206 (e) by monocytes from patients with pre-eclampsia (PE)

classified as early-onset (<34 weeks of gestation, n = 26) or late-onset (≥34 weeks of gestation, n = 59) pre-eclampsia and by monocytes from

normotensive (NT) pregnant women (<34 weeks of gestation, n = 26 and ≥34 weeks of gestation, n = 26). The results are expressed as median of

fluorescence intensity (MFI) (horizontal line), 25th and 75th percentile (box), and range (whisker). *(P < 0.05) versus NT <34 weeks; +(P < 0.05)

versus NT ≥34 weeks; #(P < 0.05) versus PE ≥34 weeks (Kruskal–Wallis test).




patients with PE. The TLR4 upregulation detected in

pre-eclamptic patients suggests that this pattern-rec-

ognition receptor plays an important role in the

exaggerated systemic inflammatory response seen in

PE.44

The association between increased endogenous

expression of TLR4 and CD64 and higher production

of TNF-a, IL-12p40, and IL-12p70 by monocytes

from patients with PE suggest that these cells can be

activated by endogenous products of maternal origin

and emphasize the participation of TLRs in the sys-

temic inflammatory response. These results imply

that the presence of an inflammatory environment

in PE can lead to monocyte polarization to an acti-

vated M1 profile, resulting in increased production

of pro-inflammatory cytokines by these cells.

Increased expression of mRNA for TLR2 and TLR4 in

neutrophils and increased production of IL-1b by

these cells, associated with higher serum levels of

TNF-a and IL-6, were observed in patients with PE,

suggesting that TLRs may modulate innate immunity

and contribute to the pathogenesis of this disease.45

On the other hand, the lower expression of

endogenous CD163 and CD206 in association with

low production of IL-10 by monocytes from pre-

eclamptic pregnant women compared with NT preg-

nant women confirms that monocytes from both

early- and late-onset PE are classically activated and

differentiated to the M1 phenotype. The expression

of classically activated monocytes or with an M1

profile is described in some diseases associated with

metabolic disorders such as type 2 diabetes33 or with

the involvement of a systemic inflammatory

response such as in sepsis.31

Although previous studies have reported an exces-

sive activation of monocytes in women with PE, as

evidenced by increased production of TNF-a, IL-6,

and IL-8 compared with NT pregnant

women13,20,44,46 and a high concentration of chemo-

kines CXCL10/IP10 in early-onset PE,12 this is the

first work that associates the expression of activation

markers and production of inflammatory cytokines

by monocyte subpopulations from early- and late-

onset pre-eclamptic pregnant women.

In conclusion, the results obtained in this study

confirm that monocytes from women with PE are

endogenously activated and polarized to an M1

inflammatory profile, by higher expression of TLR4,

CD64, and inflammatory cytokine production as well

as lower expression of surface markers of the M2

profile, CD163 and CD206, and IL-10 secretion.

Although monocytes from early-onset PE showed a

higher intensity of TLR4 expression than those from

late-onset PE, the great similarity among the several

parameters studied in both PE groups suggests that

the phenotypic characterization of monocytes cannot

be employed to differentiate these two forms of PE.

Acknowledgments

This work was supported by Fundac�~ao do Amparo �a

Pesquisa do Estado de S~ao Paulo, Brazil (FAPESP,

Grant No. 2009/11924-3).

Conflict of interest statement

The authors declare that there is no conflict of inter-

est.

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