Constraints for monocyte-derived dendritic cell functions under inflammatory conditions

Constraints for monocyte-derived dendritic cellfunctions under inflammatory conditions

Tunde Fekete1�, Attila Szabo1�, Luca Beltrame2, Nancy Vivar3,

Andor Pivarcsi4, Arpad Lanyi1, Duccio Cavalieri2, Eva Rajnavolgyi1

and Bence Rethi3

1 Department of Immunology, University of Debrecen, Debrecen, Hungary2 Department of Pharmacology, University of Firenze, Firenze, Italy3 Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, Stockholm,

Sweden4 Molecular Dermatology Research Group, Unit of Dermatology and Venerology, Department of

Medicine, Karolinska Institute, Stockholm, Sweden

The activation of TLRs expressed by macrophages or DCs, in the long run, leads to

persistently impaired functionality. TLR signals activate a wide range of negative feedback

mechanisms; it is not known, however, which of these can lead to long-lasting tolerance

for further stimulatory signals. In addition, it is not yet understood how the functionality

of monocyte-derived DCs (MoDCs) is influenced in inflamed tissues by the continuous

presence of stimulatory signals during their differentiation. Here we studied the role of a

wide range of DC-inhibitory mechanisms in a simple and robust model of MoDC inact-

ivation induced by early TLR signals during differentiation. We show that the activation-

induced suppressor of cytokine signaling 1 (SOCS1), IL-10, STAT3, miR146a and CD150

(SLAM) molecules possessed short-term inhibitory effects on cytokine production but did

not induce persistent DC inactivation. On the contrary, the LPS-induced IRAK-1 down-

regulation could alone lead to persistent MoDC inactivation. Studying cellular functions in

line with the activation-induced negative feedback mechanisms, we show that early

activation of developing MoDCs allowed only a transient cytokine production that was

followed by the downregulation of effector functions and the preservation of a tissue-

resident non-migratory phenotype.

Key words: DC . Endotoxin tolerance . IRAK-1 . TLR

Supporting Information available online

Introduction

In response to pathogen recognition or inflammatory mediators,

steady-state tissue-resident DCs exit the inflamed tissues and

transport peripheral antigens to secondary lymphoid organs,

where DCs can initiate the adaptive immune response by

triggering naıve T-cell activation. At the same time, monocytes

enter the inflamed tissues and give rise to phagocytic cells and

APCs, including DCs, thereby compensating the rapid egress of the

steady-state DC network [1–3]. The newly differentiated mono-

cyte-derived DCs (MoDCs) may act as local tissue resident APCs or

as sources of inflammatory cytokines [4, 5]. In addition, these

�These authors contributed equally to this work.Correspondence: Dr. Bence Rethie-mail: [email protected]

& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

DOI 10.1002/eji.201141924 Eur. J. Immunol. 2012. 42: 458–469Tunde Fekete et al.458

cells might obtain the ability to migrate to peripheral lymphoid

organs maintaining the activation of naıve T lymphocytes [2, 6].

Human monocytes obtain DC-like features when maintained

in culture for 5–8 days in the presence of GM-CSF combined with

IL-4 or other cytokines [7, 8]. During their differentiation

MoDCs downregulate CD14, upregulate CD1a and DC-SIGN and

obtain the ability to express CCR7 upon activation that is required

for migration towards lymphoid tissues. However, such differ-

entiation of immature MoDCs is highly unlikely to occur in

inflamed tissues where the developing cells constantly receive

stimulatory signals due to the presence of microbial compounds,

inflammatory mediators and tissue damage. It has been exten-

sively documented that long-term activation leads to functional

exhaustion of macrophages and DCs [9]. Therefore, DC inactiva-

tion by persisting stimulatory signals might counteract the devel-

opment of potent monocyte-derived APCs in the inflamed tissues.

There are several molecular mechanisms implicated in

macrophage and DC exhaustion [9, 10]. These include increased

or decreased expression of signaling components, the release of

soluble mediators that might interfere with DC functions and

altered gene expression regulation. LPS increases the suppressor

of cytokine signaling 1 (SOCS1) expression in developing MoDCs

that can inhibit NF-kB activation [11, 12] and GM-CSF signaling,

thereby interfering with MoDC survival and differentiation [11].

Chronic stimulation of MoDCs through the NOD2 molecules has

been linked with the upregulation of IRAK-M (IRAK-3), an inhi-

bitor of IRAK-1 activation [13] and IRAK-M induction has been

detected in monocytes of septic patients [14]. LPS-induced

microRNAs have been shown to act through the downmodulation

of TLR signaling components, TRAF6 and IRAK-1 via the micro-

RNA miR146a [15], IKKe via miR-155[16] in macrophages and

TAB2 via miR155 in MoDCs [17]. TLR4 expression decreased in

LPS-treated macrophages [18] and the degradation of IRAK-1 has

been linked to impaired TLR signaling in both macrophages and

DCs [19, 20]. The LPS-induced cytokine IL-10 primed IRAK1,

IRAK-4 and TRAF6 for proteasomal degradation in murine DCs

[21] and IL-10 also contributed to decreased IL-12 production via

STAT3 [22]. Although several pathways have been implicated in

the functional exhaustion of long-term activated macrophages

and DCs [9, 10], their relative contribution to the decreased

functionality is not fully understood. It is yet to be understood

whether these pathways cooperate, if they operate in different

conditions, time frames or whether the multiple inhibitory

mechanisms act in a redundant manner.

In this study we analyzed the effect of a variety of activation-

induced inhibitory factors on the cytokine production of MoDCs

that receive TLR4 stimulation early during their differentiation.

Among these, we could associate the LPS-inducible CD150 (SLAM),

STAT3, SOCS1, miR146 and IL-10 molecules with short-term

inhibitory effects on DC activation and the downmodulation of

IRAK1 as a mechanism that can contribute to persistent DC

inactivation. Early LPS treatment inactivated the MyD88-depen-

dent TLR pathways in developing MoDCs whereas TIR-domain-

containing adapter-inducing interferon-b (TRIF)-dependent gene

expressions remained intact.

We studied the effect of early activation on the functional

abilities of the developing MoDCs in order to determine whether

an inflammatory environment could allow the differentiation of

migratory MoDCs that are able to instruct T-cell responses.

Strong activation of early stage MoDCs led to inflammatory

cytokine production that was, however, not followed by the

characteristic changes of chemokine receptor expression allowing

mature DCs to migrate into peripheral lymphoid tissues. Activities

of newly developing inflammatory MoDCs might thus be limited

to peripheral tissues due to their inability to modulate chemokine

receptor expression.

Results

MoDCs are unable to upregulate inflammatory cytokinegenes when differentiated in the presence of LPS

In order to understand the mechanisms leading to impaired

functionality of chronically activated DCs we determined the

kinetics and extent of the LPS induced IL-12, TNF and IL-6 gene

expression in MoDCs developed from peripheral blood monocytes

in a 2-day culture in the presence or absence of 5 ng/mL LPS.

We used this relatively low LPS concentration as it did not

induce a strong DC activation measured at the level of inflam-

matory cytokines or the expression of CD86 and CD83 at day 2

but it consistently induced a desensitization of developing MoDCs

to further LPS-mediated activation (Fig. 1A). Thus inhibitory

signals contributing to DC inactivation may not be obscured by a

strong DC activation. We analyzed MoDC activation following a

short, 2-day culture, to better represent an in vivo situation when

monocyte precursors enter inflamed tissues and differentiate to

DCs in the presence of activation signals that readily induce

effector functions. At day 2 we observed the induction of CD1a

and CD209 (DC-SIGN) and the downregulation of CD14 on a

high proportion of developing MoDCs underlying the hypothesis

that monocytes are able to obtain DC phenotype in such short

period (Supporting Information Fig. 1).

As Fig. 1B shows, a 2-day LPS pre-treatment completely blocked

the induction of IL-12, TNF and IL-6 genes by a second LPS

stimulus whereas, without LPS pre-treatment MoDCs responded

to LPS signal with a rapid and strong induction of these genes.

To study if the tolerization of developing MoDCs by an early

encounter with stimulatory signals is a general phenomenon, or if

it is specific for single LPS stimulus, we treated the cells with a

wide variety of stimulatory factors, applied separately or in

combination with LPS between day 0 and 2 of MoDC cultures.

Few of these signals induced detectable TNF production when

applied to monocytes alone, namely, heat-killed Staphylococcus

aureus (HKSA), an inducer of TLR2 signals and CL075 that trig-

gers TLR7/8 (Fig. 1C). LPS synergistically increased the levels

of TNF when combined with CD40L, the TLR2 ligands HKSA or

Pam3Cys, with CL075 or with the combination of TNF, IL-1 and

IL-6. No activation or very low cytokine levels were observed with

TNF, IFN-g and the TLR3 ligand poly(I:C).

Eur. J. Immunol. 2012. 42: 458–469 Leukocyte signaling 459


Despite the strong initial MoDC activation induced by several

types of stimuli, when the cells were washed and reactivated by

100 ng/mL LPS at day 2, we observed a complete inhibition of

TNF production in MoDCs that differentiated in the presence of

CD40L, HKSA, Pam3Cys, CL075, TNF or the combination of TNF,

IL-1 and IL-6 (Fig. 1C, right panel). The 48 h presence of LPS

resulted in a persistent DC inactivation both when LPS was added

alone and when it was combined with any of the other activation

signals. These results showed that a wide variety of stimulatory

signals can desensitize developing MoDCs for further activation

signals and that synergistically acting stimuli do not prevent

functional exhaustion.

Contrary to other activation signals that we applied, poly(I:C)

did not tolerize MoDCs to LPS-induced activation and the pre-

treatment with IFN-g, although it did not activate DCs between

day 0 and 2, synergized strongly with a later LPS signal (Fig. 1B,

left panel).

The inability of early-stage MoDCs that develop in the

presence of various activation signals to respond to further TLR

ligation is in line with previous data obtained with macrophages

or DCs [9] and we showed here that synergistic activation signals

do not rescue the cells from functional exhaustion. In addition,

we showed the complete lack of inflammatory cytokine gene

expression in LPS-tolerized MoDCs in response to further stimuli,

suggesting a major impairment of the signaling cascade that leads

to DC activation.

LPS induces several inhibitory factors in MoDCs thatmay decrease cellular activation

In order to search for molecular mechanisms responsible for DC

inactivation by chronic stimulatory signals we compared the gene

expression pattern of MoDCs that developed for 2 days in the

Figure 1. Early stimulation of developing MoDCs induces tolerance to further activation signals. (A) MoDCs were cultured in the presence ofvarious LPS concentrations or in the absence of LPS. At day 2, the expression of CD86 and CD83 on the cell surface and the TNF concentration inthe culture supernatants were analyzed (white symbols). Alternatively, the cells were washed and reactivated using 100 ng/mL LPS and the levelsof CD86, CD83 and TNF were analyzed 1 day later (black symbols). (B) MoDCs cultured in the absence (B) or presence (& ) of 5 ng/mL LPS weretreated with 100ng/mL LPS on day 2 and the kinetics of IL-6, TNF, IL-12 p40 and p35 gene expressions were studied using real-time PCR. Meanvalues 7SD were calculated from three replicates used for each sample. (C) MoDC cultures were established in the presence of various activationsignals applied alone (open bars) or combined with 5 ng/mL LPS (black bars) and TNF concentration was measured in the supernatants at day 2(left). Alternatively, MoDCs were cultured for 2 days in the presence of various activation signals applied alone (open bars) or combined with5 ng/mL LPS (black bars), then the cells were washed at day 2 and activated with 100 ng/mL LPS for an additional day followed bymeasuring TNF concentration in the supernatants (right). Mean values 7SD were calculated from three replicates used for each sample.(A–C) Representative results of at least three independent experiments are shown.

Eur. J. Immunol. 2012. 42: 458–469Tunde Fekete et al.460


presence or absence of LPS using the Illumina microarray

technology and a TLR-pathway focused PCR array (Fig. 2A and

Supporting Information Fig. 2). Interestingly, the majority of TLR

pathway-associated genes were unaffected by the presence of LPS

measured by both technologies, suggesting no major alteration in

the expression of the pathway components required for DC

activation (Supporting Information Fig. 2). We observed a

significant upregulation of potential DC inhibitory factors in

response to 2-day exposure to LPS. These included SOCS2 and

SOCS3, known regulators of TLR pathways[12], the ITIM-

containing receptor LILRB2 implicated in DC exhaustion by CD81

suppressor T cells [23] and the molecules S100A8 and S100A9

that might inhibit DC differentiation and contribute to the

development of myeloid suppressor cells in tumor tissues [24].

The expression of CD150 (SLAM) molecules, which potently

inhibit the CD40L-induced DC activation [25], was also induced

in the presence of LPS. Other known inhibitory factors, including

ATF3, SOCS1, STAT3, TGF-b or IRAK-M, were expressed

similarly in LPS-treated or control samples. Increased gene

expression of the cytokine IL-10 was detected by PCR array in

MoDCs cultured for 2 days in the presence of LPS (2.1- to 9.5-fold

upregulation by LPS, n 5 3) and confirmed by ELISA (Fig. 2B).

Expression of miR146a and miR155 were upregulated by LPS

added at day 2 to MoDCs (Fig. 2C) in line with previous findings

[15, 16]. However miR146a levels were only minimally elevated

and miR155 was not affected in MoDCs cultured for 2 days in the

presence of LPS as compared with non-treated cells, suggesting a

time-limited functionality of these microRNAs in LPS-activated

DCs.

In order to better understand which DC modulatory factors

might participate in DC exhaustion by persistent activation

signals we analyzed the expression kinetics of a wide range of

Figure 2. LPS-induced inhibitory mechanisms in early stages of MoDC differentiation. (A) Microarray analysis of potential DC inhibitory factors inMoDCs cultured with or without 5 ng/mL LPS for 2 days. (B) IL-10 concentrations are shown in supernatants of 2-day MoDC cultures in the presenceor absence of LPS. (C) The expression of miR146a (left) and miR155 (right) was measured by real-time PCR in monocytes, MoDCs that were activatedwith 100 ng/mL LPS on day 2 of the culture for 24 h (LPS day 2), in MoDCs cultured for 3 days in the presence of 5 ng/mL LPS (LPS day 0), in MoDCsthat were cultured for 2 days with 5 ng/mL LPS and then activated with 100 ng/mL LPS (LPS day 0–2) and in MoDCs cultured without LPS (ctrl).(D) Gene expression of potential DC inhibitory molecules was analyzed using real-time PCR in MoDCs cultured with (filled squares) or without(open squares) 5 ng/mL LPS. (B–D) Data are shown as mean7SD calculated from three replicate measurements. Representative results of at leastthree independent experiments are shown.



potential inhibitory factors in MoDCs developing in the presence

or absence of LPS. As shown by Fig. 2D, gene expression of all

studied DC modulatory factors, namely, SOCS1, SOCS2, SOCS3,

IRAK-M, ATF3, S100A8 and S100A9, STAT3, LILRB2, IkBa and

IkBb and CD150 was similarly induced by the presence of LPS in

developing MoDCs showing the highest difference between LPS-

treated and non-treated MoDCs during the first day of culture.

The initial peaks in gene expression were followed by a rapid

decline in case of all of these molecules reaching the same or

minimally elevated level by day 2 in LPS-treated DCs as

compared to control cultures, supporting the microarray data

that indicated minimally altered expressions of most genes at

day 2 in response to LPS (Fig. 2A). These results might indicate a

time-limited effect of the studied molecules in DC functions

rather than a role in persistent DC inactivation.

LPS-induced SOCS1, STAT3, SLAM, IL-10 and miR146ado not inactivate DCs persistently

We set up a screening assay to study if the LPS-induced DC

modulatory molecules influence cytokine production in MoDCs.

An immediate effect of the individual factors was tested on

MoDCs that received a single activation signal on day 2 of the

culture via TLR4 or TLR7/8. A potential role in inducing long-

term DC inactivation was tested in MoDCs pre-treated for 2 days

with a low LPS dose and then activated by a second, high-dose

LPS stimulus or with CL075 on day 2 (Fig. 3A). We transfected

the monocytes with siRNAs specific for the individual DC

modulatory factors (SOCS1, SOCS2, SOCS3, STAT3, CD150,

S100A8, S100A9 and IRAK-M) or with miR146a and miR155

inhibitors, as well as with control reagents and thereafter we

Figure 3. The effect of LPS-inducible inhibitory factors on MoDC activation. (A) The LPS-induced IL-12 production of MoDCs pre-cultured in theabsence (LPS day 2) or presence of 5 ng/mL LPS (LPS day 0–2) for 2 days is shown. The tolerizing ability of an early LPS stimulus on a heterologousactivation signal was tested via comparing the CL075-induced IL-12 production of MoDCs pre-cultured in the absence (CL075 day 2) or presence of5 ng/mL LPS (LPS day 0-CL075 day 2). Alternatively, MoDCs pre-cultured or not with 5 ng/mL LPS were left without further activation (LPS day 0 or (-),respectively). The effect of IL-10 on DC activation was tested using 10 mg/mL neutralizing anti-IL10 or isotype control antibodies added at days 0and 2. The effect of STAT3, SOCS1, SOCS2, SOCS3, S100A8, S100A9, IRAK-M and CD150 molecules was tested by transfecting the monocyteprecursors with siRNA molecules targeting mRNA of the individual molecules. The miR146a and miR155 effects on IL-12 production were analyzedby transfecting the monocytes with specific locked nucleic acid (LNA) miRNA inhibitors or with LNA miRNA control inhibitor. Data are presentedas mean7SD calculated from three replicate measurements. Representative results of at least three independent experiments are shown.(B) Monocytes were transfected with miR146a or miR155 precursor molecules or with control miRNA. Thereafter MoDC cultures were establishedand maintained for 2 days. The cells were activated using LPS (100 ng/mL), CD40L-expressing L cells (1:10 L cell:DC ratio), poly (I:C) (20mg/mL) orCL075 (1 mg/mL) for 24 h. The effect of miRNA molecules on cytokine production is compared with control miRNA-transfected samples. Cytokineproduction induced by the different activation signals was tested in three to five different experiments (the effect of microRNAs was not calculatedin samples where cytokine levels did not reach the detection limit of the ELISA).



cultured the cells for 2 days in the presence or absence of LPS. We

studied the role of LPS-induced IL-10 production in DC

inactivation using IL-10-specific neutralizing antibodies included

during LPS-pre-treatment as well as during reactivation of the

cells. At day 2, we activated both LPS pre-treated and non-treated

cells with LPS or CL075 and we measured IL-12 production. We

selected siRNA reagents for this assay that could induce an at

least three-fold decrease in the mRNA levels of the individual

genes by day 2 in both LPS pre-treated and non-treated MoDCs

(data not shown) assuming that such inhibitory effect on the

mRNA levels may efficiently counteract the LPS-induced upregu-

lation of the different inhibitory factors (Fig. 2).

As shown on Fig. 3A, MoDC transfection by siRNAs that

targeted STAT3, CD150 or the inhibition of miR146a and IL-10

increased IL-12 production by the cells that received a single

activation by LPS or CL075 at day 2. Transfection with SOCS1-

specific siRNA led to increased IL-12 production induced by LPS

at day 2 without affecting the activation induced by CL075. These

inhibitory factors, when induced during MoDC activation, may

act as immediate negative regulators that might help to terminate

gene expression in activated DCs.

To further test a potential inhibitory function of miR146a and

miR155 on MoDC activation, we transfected monocytes with

precursors of these miRs and activated the cells using LPS,

poly(I:C), CL075 or CD40L after 2 days of culture (Fig. 3B). In

line with the data obtained with miR146a-specific siRNAs,

transfection of developing MoDCs with miR146a led to decreased

IL-12 and TNF production in response to all tested activation

signals. Transfection with miR155 inhibitor led to decreased

IL-12 producing ability (Fig. 3A) and, similarly, transfection of

MoDCs with miR155 led to a mild, but consistent, decrease of

IL-12 and TNF production (Fig. 3B). These results possibly reflect

multiple, often counteracting, effects of miR155 on DC activation

pathways that is also indicated by previously described effects of

this miR, both stimulatory or inhibitory, on macrophage and DC

functions [16, 17, 26].

Downregulation of SOCS2, SOCS3, IRAK-3, S100A8 and

S100A9 led to unaffected or decreased IL-12 production, indicating

no inhibitory effect of these factors in MoDC activation (Fig. 3A).

Importantly, inhibition of none of the tested DC modulatory

molecules had an impact on the strong inhibitory effect of the LPS

pre-treatment on IL-12 production triggered by a second activa-

tion signal (Fig. 3A). MoDC activation early during differentia-

tion may thus lead to functional exhaustion independently of the

tested regulatory factors.

LPS-induced IRAK1 downregulation is sufficient toinhibit further activation

TLR4 and IRAK1 proteins are degraded in response to long-term

LPS triggering in macrophages and in DCs [18–20] whereas the

inhibitory protein IRAK-M can be upregulated upon chronic DC

activation [13]. We compared TLR4 expression in MoDCs

developing with or without 5 ng/mL LPS for 2 days using flow

cytometry or Western blot and found no sign of decreased TLR4

expression in the presence of LPS (data not shown). Thereafter

we studied IRAK-1 and IRAK-M protein levels in MoDCs

developing in the presence or absence of LPS using western blot

and we detected the downregulation of IRAK1 by day 2 in the

presence of LPS (Fig. 4A). IRAK-M levels slightly decreased as

well, indicating, together with our data obtained with the IRAK-M-

specific siRNA (Fig. 3A), that an upregulation of IRAK-M might

not stand as the mechanism underlying MoDC endotoxin tolerance.

In order to determine whether decreased IRAK-1 levels could play

an important role in DC inactivation, we transfected developing

MoDCs with IRAK1-specific siRNA. As shown on Fig. 4B, decreased

IRAK-1 expression resulted in low IL-12 production when MoDCs

were activated on day 2 by LPS or CL075. These results indicate

that the activation-induced IRAK1 downregulation might play an

important role in the functional exhaustion of MoDCs as this event

alone can lead to decreased cytokine production by activated DCs.

LPS-tolerized MoDCs shift from Myd88 to TRIF-dependent signaling pathways

Previous studies have indicated a developmental blockade in

MoDC differentiation in response to persistent TLR activation

[11, 27, 28] or an impaired TLR signaling as the underlying

mechanism for LPS-induced tolerance [9, 10, 14, 15, 20, 21]. To

better understand how an early activation influences MoDC

functionality we studied survival, differentiation and signaling

abilities of MoDCs developing in the presence of LPS.

Figure 4. Downregulation of IRAK-1 in MoDCs developing in thepresence of LPS. (A) IRAK-M and IRAK-1 expression was studied inmonocytes or in MoDCs developing in the presence of 5ng/mL LPS for 1or 2 days using western blot. The effect of LPS on IRAK-1 and IRAK-Mprotein levels is shown in the lower panel calculated from the bandintensities measured in three independent experiments. (B). Mono-cytes were transfected with IRAK-1-specific or control siRNA moleculesand then MoDC cultures were established and maintained for 2 days.The cells were activated for 24 h using LPS (100 ng/mL) or CL075(1 mg/mL) and IL-12 concentration was measured in the supernatants.Data are shown as mean7SD calculated from three replicate measure-ments. A representative result of three independent experiments isshown.



As shown in Fig. 5A, TLR ligands, TNF or CD40L had a variable

effect on MoDC differentiation by day 2 and none of the stimuli led

to a substantial increase in apoptosis. Ligation of TLR2 by zymosan,

or HKSA and the TLR7/8 ligand CL075, led to the retention of high

CD14 expression on a subset of cells and blocked CD1a expression.

Other signals, however, did not have a major impact on MoDC

differentiation markers despite their ability to decrease the sensi-

tivity to further activation (Fig. 1). Monocyte activation may thus

prevent DC differentiation in the case of some particular TLR

ligands; however, such effect does not fully overlap with the toler-

izing ability of the different stimuli.

In order to identify which TLR-induced signaling pathways

are impaired in MoDCs that received an early LPS stimulation we

studied MAPK, NF-kB and IRF-3 activations in these cells. Acti-

vation of MAPKs is attributed to signals transmitted by the

Myd88-dependent arm of the TLR pathways that might be

particularly affected by the downmodulation of IRAK-1. Accord-

ingly, LPS-induced phosphorylation of the Erk1/2 and p38

kinases, as well as phosphorylation of CREB/ATF-1 transcription

factors, often occurring via p38 activation, were abrogated by

LPS pre-treatment of developing MoDCs (Fig. 5B). On the

contrary, DCs differentiating in the absence of LPS responded

Figure 5. Early activation of MoDCs modulates differentiation and TLR signaling. (A) Survival and the expression of the differentiation markersCD1a, CD14 and CD11c were measured in MoDCs cultured in the presence of various activation signals for 2 days. (B) MoDCs were cultured in thepresence (open squares) or absence (black diamonds) of 5 ng/mL LPS for 2 days. Thereafter the cells were activated with 100 n/mL LPS and thephosphorylation of Erk1/2, p38 MAP kinases and the transcription factors CREB/ATF1 were studied by flow cytometry. (C) NF-kB signaling wasstudied in LPS-activated MoDCs that were cultured for 2 days in the presence or absence of 5 ng/mL LPS. IkBa levels, phosphorylation of the IkBaand p65 (on serine 529, 276 and 536) were analyzed by western blot. (D) IRF3 phosphorylation in response to LPS (top) or poly(I:C) (bottom) wasstudied by western blot in MoDCs cultured for 2 days in the presence or absence of 5ng/mL LPS. (E) IFN-b gene expression in response to poly(I:C)activation was studied by real-time PCR in day-2 MoDCs pre-cultured or not with 5 ng/mL LPS (left). Mean1SD values were calculated from threereplicate measurements. In similar assays we compared the induction of the IFNa1, IFNa2, CCL5 and CXCL10 genes in response to poly(I:C)between LPS pre-treated and non pre-treated MoDCs (right). Mean1SD values were calculated from four independent experiments. In all otherpanels representative results of at least three independent experiments are shown.



readily with Erk1/2, p38 and CREB/ATF-1 phosphorylation to

LPS stimulation.

The primary step of NF-kB activation is the phosphorylation-

dependent degradation of the IkB components, a prerequisite for

NF-kB nuclear translocation [29]. Interestingly, LPS-induced

IkBa phosphorylation occurred similarly in LPS pre-treated and

control MoDCs and we did not detect a different level of the total

IkBa protein in these samples either (Fig. 5C). These results

indicate that NF-kB might be activated by TLR-dependent signals

in LPS-tolerized MoDCs. Further activity of NF-kB is tuned by

enzymatic modifications that include phosphorylation at multiple

residues. The NF-kB subunit p65 is phosphorylated at S276 in

order to gain strong transcriptional activity, whereas its functions

are further modulated by phosphorylations at other sites of the

protein [30]. We found a similar S276 and S536 phophorylation

in response to LPS in both LPS pre-treated and control MoDCs

(Fig. 5C). S529 phosphorylation was, on the other hand, inhib-

ited in LPS-pretreated DCs, indicating a partial impairment of

NF-kB regulation following persistent LPS signals. However, func-

tional significance of S529 phosphorylation is not known.

The partial activation of NF-kB in spite of the decreased

Myd88-dependent signal transduction might indicate functional

MyD88-independent, TRIF-dependent signal routes. Indeed, we

found a strong IRF-3 phosphorylation in response to TLR3 or

TLR4 ligation by poly(I:C) and LPS, respectively, in both LPS-

pretreated and control MoDCs (Fig. 5D). IRF-3 phosphorylation

was rather elevated in LPS–pre-treated cells (3.8- and 2.1-fold

higher than in non-LPS–pre-treated cells when using 2-h LPS or

poly(I:C) stimulation respectively, as calculated from the densi-

tometry analysis of three independent experiments) and the

increased IRF-3 activation was accompanied by higher IFN-bexpression in LPS pre-treated MoDCs as compared with control

cells (Fig. 5E). Similar to the observed effect on IFN-b, poly(I:C)

induced higher expression of other genes sensitive for TRIF-

dependent regulation (IFN-a1, IFN-a2 and CCL5) when the cells

received LPS pre-treatment whereas we did not consistently

observe a similar effect on CXCL10 expression. Overall, our

results indicated the downregulation of MyD88-dependent TLR

signals in response to LPS pre-treatment of developing MoDCs.

The TRIF-dependent TLR pathways, on the other hand, might

remain functional following a persistent LPS stimulation.

MoDCs activated early in development have a limitedability to modulate chemokine receptor expression

We compared gene expression of chemokine molecules in MoDCs

cultured with or without LPS for 2 days and observed a significant

increase in the expression of CCL5, CCL18, CCL19, CCL23,

CCL24, CCL26, CXCL1, CXCL2 and CXCL5, in the presence of LPS

that suggests an increased ability of the LPS-treated MoDCs to

attract both resting and activated T cells, as well as granulocytes

(Fig. 6A). In addition to such possible contribution to the cellular

influx associated with tissue inflammation, LPS-treated MoDCs

might increase their motility by cleaving extracellular matrix

constituents as suggested by the elevated MMP7, MMP9 and

MMP12 mRNA levels in these cells.

In order to understand whether MoDCs that received activa-

tion signals at early stages of their development could obtain

migratory potential towards lymphoid tissues, and contribute to

naıve T-cell activation, we studied their chemokine receptor

pattern during the first day of culture in the presence of a wide

range of activation stimuli. As MoDCs responded readily with a

strong cytokine production when receiving activation signals

Figure 6. Early activation of developing MoDCs induces chemokineproduction but no change in CCR7 and CCR5 cell surface expression.(A) Microarray data shows gene expression of chemokines, chemokinereceptors and matrix metalloproteinases in developing MoDCsextracted from the complete list of differentially expressed genes.Monocytes were isolated from five different blood donors and werecultured in the presence (black diamonds) or absence (open diamonds)of LPS for 2 days. (B) Expression of CCR5 (top) and CCR7 (bottom) wasanalyzed in MoDCs using flow cytometry at day 1 (left) or day 6 (right)following the activation of cells using a range of TLR ligands, cytokinesor CD40L alone (white bars), or in combination with LPS (white andblack bars). Representative results of three independent experimentsare shown.



(day 1) and became later functionally exhausted and incapable to

respond to further stimulation (day 2), an early chemokine

receptor modulation might be prerequisite for the migration of

functional, not-exhausted MoDCs to lymphoid tissues. We studied

CCR5 and CCR7 expression on MoDCs that received activation

signals during the first day of their culture and compared

these cells with MoDCs that received the same activation signals

at a more differentiated stage, at day 5 of the culture. Interestingly,

CCR5, a chemokine receptor that primes migration to inflamed

peripheral tissues, was not downregulated and CCR7 was not

induced when MoDCs received activation signals early in their

development. On the contrary, MoDCs that developed for 5 days

without activation downregulated CCR5 in response to LPS,

HKSA, zymosan or CD40L and several of the tested activation

signals induced the expression of CCR7 on these cells (Fig. 6B).

These results showed that the inability of MoDCs to modulate

their chemokine receptors early during their differentiation might

limit egress from peripheral tissues and predispose these cells to

short-term inflammatory functions in the periphery. Longer

differentiation, free of activation signals, might be required for

the acquisition of a migratory phenotype in response to later

activation; however, such differentiation pattern may not occur

in inflamed tissues.

Discussion

Persistent macrophage and DC activation by TLR ligands leads to

particularly powerful inhibitory mechanisms blocking further

activation by the same or heterologous stimuli [9]. There are

several inhibitory factors induced in response to TLR stimulation; it

is still unclear, however, how these factors contribute to tolerance

for further activation. Some pathways have been connected, like

miR146a and IL-10 might both contribute to decreased IRAK1

expression [11, 21], but the present view supports several coexisting

inhibitory pathways in activated DCs and macrophages. Whether

these pathways are redundant, additive or synergistic or act in

different conditions or time frames is yet to be understood. Since

DCs developing from monocyte precursors in the inflamed tissues

might be particularly affected by the constant presence of microbial

compounds and inflammatory mediators, we decided to study

which inhibitory pathways are activated in MoDCs in the presence

of early and persistent TLR4 stimulation. We set up an assay

distinguishing a timely separated role for the different inhibitory

molecules and showed that the LPS-induced SOCS1, STAT3, SLAM,

miR146a and IL-10 molecules possessed an immediate effect

decreasing the activation induced IL-12 production. None of these

molecules, however, played an essential role in the establishment of

tolerance to further activation signals. The short-term influence of

the tested inhibitory signaling components was probably a

consequence of the transient increase in their gene expression or

the presence of other, more efficient inhibitory pathways. Although

not tested here, it is also possible that certain inhibitory factors

could modulate the expression of particular genes in DCs, thereby

inducing a qualitative tuning of cellular functions. Contrary to these

pathways, IRAK-1 downregulation, occurring in MoDCs receiving

early activation through TLR4 during differentiation, might alone be

sufficient to inhibit further activation through TLR molecules, as

demonstrated by the strong inhibitory effect of a siRNA induced

IRAK-1 downregulation on IL-12 secretion.

Previously, SOCS1 has been implicated in establishing toler-

ance in MoDCs that developed in the presence of TLR4, TLR2 or

TLR3 ligands through inhibiting GM-CSF receptor signaling and

thereby preventing DC differentiation [11]. A blockade of the DC

differentiation pathway as a consequence of TLR stimulation on

monocyte precursors has also been indicated by other studies, in

case of human MoDCs in vitro [27] and in monocytes entering the

skin in response to Gram-negative bacteria [28]. On the other hand,

several studies indicated impaired TLR pathways in persistently

activated macrophages and DCs as the underlying mechanism for

their decreased functionality. We showed IRAK-1 downregulation

and decreased MyD88-dependent signaling activity in response to

early LPS activation in MoDC development in the absence of any

detectable change in the survival rate. Some activation stimuli,

including zymosan, HKSA or CL075, inhibited the upregulation

of CD1a and the downregulation of CD14 on a subset of the

developing MoDCs by day 2. Other factors, like PAM3Cys, TNF

or CD40L had, on the other hand, no effect on phenotypic

MoDC differentiation although these molecules were able to

induce a functional MoDC exhaustion. Although both mechanisms

might operate, downmodulation of TLR pathway intensity during

early MoDC activation might induce tolerance to further activation

irrespective of the differentiation stage of the cells.

SOCS1 upregulation, however, represents a potent negative

feedback mechanism that can decrease DC activation, as

demonstrated by our results showing higher IL-12 production in

LPS-activated DCs following SOCS1 downregulation and also by

the increased Th1-type T-cell responses induced by DCs of

SOCS1�/� mice [31]. SOCS1 might directly interfere with NF-kB

activation [32] or it can contribute to the degradation of the

adapter protein Mal, associated to TLR4 and TLR2 [33]. The

several inhibitory mechanisms suggest that SOCS1 could most

probably influence DC activation not only through blocking DC

differentiation. Indeed, Mal modulation might explain why

SOCS1 downregulation increased TLR4-mediated activation but

did not affect the IL-12 production triggered by a ligand for TLR7

and TLR8, receptors that do not utilize Mal. Nevertheless, our

results showed no effect of SOCS1 downregulation on the

permanent inactivation of MoDCs that developed in the presence

of continuous TLR ligation, indicating that the LPS-induced

SOCS1 molecules act as short-term inhibitory factors.

Most studies on macrophage or DC inactivation by persistent

TLR stimulation have been limited to in vitro conditions. Endo-

toxin tolerance of monocytes has been described in septic

patients [14, 34]; however, a broader significance of macrophage

and DC exhaustion in response to persistent activation signals is

still unknown.

MoDCs might be affected by the inhibitory signals originated

from constant activation when differentiating in inflamed tissues.

A recent study showed a very rapid DC differentiation of peripheral



blood monocytes followed by their lymph node homing in mice that

received LPS injections [6]. Circulating monocytes might thus

differentiate into migratory DCs within a time frame short enough

to preserve their full functionality. Such rapid differentiation was

not observed when ligands for other TLRs were injected, suggesting

that the migratory DC differentiation from blood monocytes

might be a mechanism specifically triggered by Gram-negative

bacteria. Other data, however, showed that intradermal injection

of Gram-negative bacteria or LPS to mice blocked the differ-

entiation of migratory DCs from monocyte precursors [28]. These

data suggest that migratory DC differentiation in the peripheral

tissues might be impaired if the activation signals reach the

monocyte precursors before their commitment to the DC differ-

entiation pathway. Our data support this hypothesis by showing

that activation of early MoDC precursors leads to inflammatory

cytokine and chemokine production but the cells, at early stage of

DC differentiation, have a limited ability to modulate their

chemokine receptor expression required for lymph node homing.

The cytokine producing ability of the developing inflammatory

MoDCs can be terminated by the functional exhaustion before the

cells differentiate to mature DCs capable of reprogramming their

chemokine receptor profile. Early activation of developing MoDCs

may thus set the threshold of DC migration to LNs, thereby limiting

the continuous transfer of inflammatory signals to T lymphocytes.

Materials and methods

DC cultures

Monocytes were isolated from buffy coats by Ficoll gradient

centrifugation (Amersham Biosciences) and magnetic cell separa-

tion using anti-CD14–conjugated microbeads (Miltenyi Biotech).

The purified cells were cultured at a density of 2� 106 cells/mL in

RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO) supplemen-

ted with 10% FCS (Invitrogen), 75 ng/mL GM-CSF (Gentaur) and

100 ng/mL IL-4 (Peprotech EC). For DC activation we used the

TLR ligands LPS, CL075, HKSA, Zymosan, Pam3Cys or poly(I:C),

all from Invivogen or soluble CD40L, INF-g, TNF, IL-1 or IL-6

from Peprotech. Polyclonal IL-10 neutralizing antibodies and the

goat isotype control Abs were purchased from R&D Systems.

Microarray analysis

RNA was isolated from MoDCs precultured with or without

5 ng/mL LPS for 2 days using TRI reagent (Invitrogen) followed

by a second purification on RNeasy coloumns coupled with

DNase I treatement (Qiagen) according to the manufacturer’s

recommendations. The extracted samples were labeled with Cy5,

hybridized on Illumina Whole Genome HT12 microarrays, accord-

ing to the manufacturer’s instructions. After scanning, bead-level

data was converted into bead-summary data using the Illumina

BeadStudio software. Prior to normalization, array probes were

annotated using their sequence and converted to unique nucleotide

identifiers (nuIDs). Background signal was assessed and corrected

using the intensity signal from the control probes present on the

array, and then quantile normalization was performed with the aid

of the lumi R package [35]. Microarray data has been submitted

to the Array Express repository (accession number: E-MTAB-658).

Differentially expressed genes were calculated using the Rank

Product algorithm [36]. differentially expressed genes were called

significant when their corrected p-value (percentage of false

positives) was equal to or lower than 0.05.

Real-time PCR

Total RNA was extracted using TRIreagent (Invitrogen) and

cDNA synthesis was performed using the High Capacity cDNA

Reverse Transcription Kit of Applied Biosystems. All gene

expression assays were purchased from Applied Biosystems.

Results were normalized with the expression of the housekeeping

gene cyclophilin or with RNU48 in case of the miR assays. The

expression level of these genes did not vary between the cell types

or treatments used in our experiments. PCR was performed using

the ABI7900 Real-Time PCR system (Applied Biosystems). TLR

focused PCR array was purchased from Qiagen and used

according to the manufacturer’s recommendations.

Flow cytometry

The FITC-labeled anti-CD14 and anti-CD86, PE-labeled anti-

CD1a, PE-Cy5 conjugated anti-CD83, allophycocyanin-labeled

anti-CD11c and Annexin V were purchased from BD Pharmingen,

the fluorescein-conjugated anti-CCR7 antibody from R&D

Systems. Fluorescence intensities were measured with FACSort

(Becton Dickinson) and data analyzed with FlowJo v. 8.4.4

software (Tree Star).

DC transfections

Gene-specific siRNA reagents were purchased from Applied

Biosystems (STAT3, SOCS1, S100A8, S100A9), Dhramacon

(IRAK-M) or from Invitrogen (SOCS2, SOCS3, IRAK-1, CD150)

with the appropriate non-targeting control RNAs obtained from the

same companies. The microRNA LNA-inhibitors for miR146a and

miR155 or the control LNA-inhibitor were purchased from Exiqon.

Precursors for miR146a and miR155 as well as non-targeting

microRNA controls were purchased from Applied Biosystems.

Transfections were performed in Opti-MEM medium (Invitrogen)

in 4-mm cuvettes (Bio-Rad) using GenePulser Xcell (Bio-Rad).

Cytokine concentrations

IL-12 and TNF production was analyzed in culture supernatants

using ELISA (BD Pharmingen) according to manufacturer’s

recommendations.



Western blotting of DC protein lysates

Protein extraction was performed by lysing cells in Laemmli buffer

(0.1% SDS, 100 mM Tris, pH 6.8, bromophenol blue, 10% glycerol,

5% v/v b-mercaptoethanol). Proteins were denaturated by boiling

for 10 min. Samples were separated by SDS-PAGE using 7.5–10%

polyacrylamide gels, and transferred to nitrocellulose membranes.

Non-specific binding was blocked by TBS-Tween-5% non-fat dry

milk for 1 h at room temperature. Anti-IRAK-1, anti-IRAK-M, anti-

IRF3, anti-pIRF3, anti-IkBa, anti-pIkBa, anti-pp65-S276, anti-

pp65-S536 (Cell Signaling, Danvers, MA, US), anti pp65-S529

(Santa Cruz, CA, US) and anti-b-actin antibodies (Sigma-Aldrich)

were used at a dilution of 1:1000; secondary antibody (GE

Healthcare, Little Chalfont Buckinghamshire, UK) was used at

1:5000. Membranes were washed three times in TBS-Tween; then

incubated with anti-rabbit conjugated to horseradish peroxidase

for 30 min at room temperature. After three washes with TBS-

Tween, protein samples were visualized by enhanced chemilu-

minescence (SuperSignal West Pico Chemiluminescent Substrate;

Thermo Scientific, Rockford, IL, USA).

Acknowledgements: This work was supported by the Swedish

Medical Research Council, by the Hungarian Scientific Research

Fund (72532), the DC-THERA and the FP7 Tornado-222720 program.

Conflict of interest: The authors declare no financial or

commercial conflict of interest.

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Abbreviations: HKSA: heat-killed Staphylococcus aureus � LNA: locked

nucleic acid � MoDC: monocyte-derived DC � SOCS: suppressor of

cytokine signaling � TRIF: TIR-domain-containing adaptor-inducing

interferon-b

Full correspondence: Dr. Bence Rethi, Department of Microbiology,

Tumor and Cell Biology, Karolinska Institutet, Nobels vag 16,

Stockholm 17177, Sweden

Fax: 146-8330498

e-mail: [email protected]

Additional correspondence Prof. Eva Rajnavolgyi, Department of

Immunology, University of Debrecen, Egyetem ter1, Debrecen 4032,

Hungary

Fax: 13652417159

e-mail: [email protected]

Received: 6/7/2011

Revised: 30/9/2011

Accepted: 26/10/2011

Accepted article online: 7/11/2011



Constraints for monocyte-derived dendritic cell functions under inflammatory conditions

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Transcript of Constraints for monocyte-derived dendritic cell functions under inflammatory conditions