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The differential response of human dendritic cells tolive and killed Neisseria meningitidis

Hannah E. Jones,1,2 Heli Uronen-Hansson,3

Robin E. Callard,2 Nigel Klein1 and

Garth L. J. Dixon1,4*1Infectious Diseases and Microbiology Unit and2Immunobiology Unit, Institute of Child Health, UCL,

London, UK.3Department of Experimental Medical Science,

Immunology Lund University, Lund, Sweden.4Department of Microbiology, Camelia Botnar

Laboratories, Great Ormond Street Hospital, London,

UK.

Summary

There is currently no effective vaccine for Neisseria

meningitidis (Nm) serogroup B. Generation of

optimal immune responses to meningococci could

be achieved by targeting meningococcal antigens to

human dendritic cells (DCs). Recent studies have

shown that diverse DC responses and subsequent

generation of protective immunity can be observed if

the microbes are viable or killed. This is important

because the host is likely to be exposed to both live

and killed bacteria during natural infection. There are

currently few data on comparative DC responses to

live and killed meningococci. We show here that

exposure of human DC to live meningococci does

not result in a typical maturation response, as deter-

mined by the failure to upregulate CD40, CD86,

HLA-DR and HLA-Class I. Despite this, live meningo-

cocci were potent inducers of IL-12 and IL-10,

although the ratios of these cytokines differed from

those to killed organisms. Our data also suggest that

enhanced phagocytosis of killed organisms com-

pared with live may be responsible for the differential

cytokine responses, involving an autocrine IL-10-

dependent mechanism. The consequences of thesefindings upon the effectiveness of antigen presen-

tation and T-cell responses are currently under

investigation.

Introduction

Neisseria meningitidis (Nm) remains a major cause of

bacterial meningitis and septicaemia worldwide. While

conjugate capsular vaccines have been developed suc-

cessfully against serogroups A, C, W135 and Y, the devel-

opment of a safe and effective vaccine against serogroup

B bacteria has remained elusive (Jodar et al., 2002; Har-

rison, 2006). Use of serogroup B polysaccharide capsule

as a vaccine is regarded as being problematic due to its

poor immunogenicity and concerns over safety due to the

similarity of group B polysialic acid capsule to human

neural cell adhesion molecules (Finne et al., 1983). Thecurrent serogroup B vaccines are based on subcapsular

outer membrane antigens; however, their efficacy has

been found to be limited especially in infants, who are at

most risk of the disease (Bjune et al., 1991; Cartwright

et al., 1999; Tappero et al., 1999). Although a number of

other promising vaccine candidates (Pizza et al., 2000;

Giuliani et al., 2006) have been identified, there are

limited data on how these should be presented to the

immune system in order to induce immunity to a broad

range of meningococcal antigens, elicit long-term memory

and provide protection for all age groups. Understanding

how meningococci interact with dendritic cells (DCs) islikely to be key to achieving these aims.

Dendritic cells are the key orchestrators of the immune

responses to the microbial world (Banchereau and Stein-

man, 1998). Immature DCs in the periphery and submu-

cosa sample the external environment and capture

antigen, including whole bacteria, after which they migrate

to secondary lymphoid tissue where they present pro-

cessed antigen to stimulate antigen-specific T-cells

(Banchereau et al., 2000). The outcome of the interaction

between DCs and lymphocyte is critically influenced by

the release of both cytokines and chemokines and by the

expression and function of co-stimulatory molecules by

DCs. The nature of the DC response to microbial encoun-

ter appears to be dictated to a large extent by the

presence of pathogen-associated molecules, such as

lipopolysaccharide (LPS) present on the microbe, and the

signals generated as a result of the interaction of these

microbial components and host receptors, principally toll-

like receptors (TLRs) (Pulendran, 2005).

We have previously shown that both the presence and

structure of LPS in the outer membrane of Nm are critical

Received 12 March, 2007; revised 30 May, 2007; accepted 6 June,2007. *For correspondence. E-mail [email protected]; Tel.(+44) 207813 8594; Fax (+44) 207813 8494.

Cellular Microbiology (2007) 9(12), 28562869 doi:10.1111/j.1462-5822.2007.01001.xFirst published online 10 July 2007

2007 The AuthorsJournal compilation 2007 Blackwell Publishing Ltd
mailto:[email protected]:[email protected]


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in defining the response of human DCs to the bacteria.

Wild-type killed meningococci induce both DC maturation

and production of cytokines, particularly IL-10 and bioac-

tive IL-12 p70, whereas neither purified LPS nor LPS

deficient meningococci, even with exogenously added

LPS, are able to induce high levels of these cytokines

(Dixon et al., 2001). The reason why wild-type meningo-

cocci are capable of inducing high levels of IL-10 and

IL-12 appears to stem from the requirement of LPS within

the outer membrane for efficient internalization by human

DCs (Uronen-Hansson et al., 2004a).

It is clear, however, that both DC maturation and pro-

duction of certain cytokines (particularly IL-6) are inde-

pendent of phagocytosis, and moreover can be induced in

the absence of LPS (Uronen-Hansson et al., 2004a). For

example, it has been shown that major meningococcal

outer membrane proteins porA (Al Bader et al., 2004) and

PorB (Singleton et al., 2005) can induce both DC matu-

ration and the production of certain cytokines. Therefore,

in addition to being target antigens these outer membrane

components also appear to possess adjuvant activitywhich could be advantageous for their use as vaccines.

Such preparations, however, do not appear to support

significant production of IL-10 or bioactive IL-12 by DCs,

both considered to have a critical influence on T-cell

responses (Al Bader et al., 2004). Targeting DCs by uti-

lizing whole bacteria through the phagocytic pathway has

the advantage that it is a highly efficient route for antigen

delivery. Once within the DC, LPS and other bacterial

components such as porins in the bacteria then can

engage with a variety of receptors, including TLRs,

located within endosomal and phagosomal compartments

(Underhill et al., 1999; Underhill and Ozinsky, 2002;Uronen-Hansson et al., 2004b). It may be that the signals

generated by these processes are critical in induction of

T-cell polarizing cytokines.

As immune responses to live and killed organisms can

differ markedly (Kikuchi et al., 2004; Skinner et al., 2004;

Rey-Ladino et al., 2005), we sought to compare the

response of DC to live and killed Nm. We postulated that

live organisms would be at least as effective as killed

counterparts at inducing DC maturation and cytokine

production. Surprisingly, we found that live Nm were inca-

pable of inducing full DC maturation as compared with

their killed counterparts. Despite the lack of a typical

maturation response, live Nm could induce both IL-12

production and IL-10 by DCs, although the ratios of these

two cytokines were quite different compared with the

response to killed organisms. The results presented here

suggest that live meningococci may specifically modulate

DC function which could have important influences on the

nature of T-cell and B-cell responses that occur during

natural infection/carriage and may have implications in

the design of novel vaccines against this pathogen.

Results

A distinct surface phenotype is observed when DCs are

exposed to live Nm compared with killed bacteria

We have previously shown that killed Nm increase

surface expression of co-stimulatory molecules CD86,

CD40, CD83, HLA-Class I and HLA-DR on human DCs,

consistent with their maturation and activation (Dixon

et al., 2001). We first sought to determine whether live Nm

were able to induce similar DC phenotypic changes. DCs

were incubated for 1824 h with either live or killed Nm at

a multiplicity of infection (moi) ranging from 1 to 100 and

expression of these activation markers determined by

flow cytometry. Unexpectedly, there was no increase in

surface expression of CD40, CD86 and MHC Class I

when DCs were cultured with live Nm (Fig. 1A). This

observation was independent of the donor from whom the

DCs were derived and was consistent over a range of

1100 moi (Fig. 1B). A modest increase in expression of

HLA-DR and CD83 was observed in response to live Nm

(Fig. 1A) suggesting that these cells were undergoingsome maturation. The possibility that the induction of

co-stimulatory molecules expression in response to live

Nm was delayed compared with killed bacteria was

explored. The differential pattern of DC cell surface

expression between live and dead meningococci was the

same at 48 h as observed at 24 h (data not shown).

We investigated the possibility that these observed dif-

ferences could be due to excessive DC necrosis or apo-

ptosis in co-culture with proliferating bacteria. Despite the

exposure of DCs to continually proliferating bacteria,

there was no difference in the proportion of dead DCs

(ranging from 5% to 15%) for all culture conditions asdetermined by trypan blue staining. To address the pro-

portion of DCs that were undergoing apoptosis, we mea-

sured caspase activity by flow cytometry. Figure 2 shows

the percentage of DCs positive for caspase activity follow-

ing stimulation with medium, live and killed Nm (100 moi).

There is a modest increase in apoptotic cells after activa-

tion with live Nm but still less than 24% compared with

14% with dead bacteria. This amount of death is not

sufficient to explain the inability of live bacteria to induce

CD40 and CD86 expression. We conclude from these

data that the observed differences between proliferating

and killed bacteria are unlikely to be due to excessive

cellular necrosis or apoptosis in live co-cultures.

Brief exposure to dividing bacteria determines the

distinct maturation response of DCs to live Nm

We next investigated if bacterial proliferation and protein

synthesis was necessary for the distinct DC surface mol-

ecule expression pattern we had already observed in

response to livebacteria. To achieve this, live bacteria were

Dendritic cell response to Neisseria meningitidis 2857

2007 The AuthorsJournal compilation 2007 Blackwell Publishing Ltd, Cellular Microbiology, 9, 28562869


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exposed to bacteriostatic levels (2.510 mg ml-1) of

chloramphenicol (cm) prior to and during co-culture with

DCs. Cm inhibits both microbial protein synthesis by

binding to the 50S subunit of the ribosome and impairing

peptidyl transferase activity, and is rapidly but rever-

sibly bacteristatic. In this culture system, the concentra-

tions of antibiotic used completely inhibit meningococcal

proliferation, but do not alter viability (see Experimental

procedures).

Our results showed that when cm is added prior to

co-culture with DCs, the pattern of CD40, CD83, CD86,

HLA-Class 1 and HLA-DR expression was identical to that

seen with DCs exposed to killed Nm (Fig. 3A). This was

consistent among all donors tested and was observed at

all starting concentrations of bacteria (moi of 1100). We

then investigated over what time period DCs have to be

exposed to proliferating meningococci to result in the dis-

tinct DC maturation response. When cm was added after

6 h exposure to live bacteria, the pattern of cell surface

molecule expression was similar to that seen with live

bacteria. This is illustrated in Fig. 3B using CD40 expres-

sion as an example. Cm had no effect on DC maturation

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Fig. 3. Bacteristatic doses of chloramphenicol restore capacity of live Nm to induce DC maturation.A. DCs were stimulated with live or killed Nm (moi 100) with and without cm (5 mg ml-1) for 18 h and expression of CD40, CD83, CD86,HLA-Class 1 and HLA-DR determined by flow cytometry.B. A delay in addition of chloramphenicol to live meningococcal/DC co-culture by 6 h results in no induction of CD40 expression.The data shown here are representative of experiments from five separate donors.




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when added alone, nor did it alter the induction of matu-

ration markers in response to either killed bacteria or LPS

(data not shown). These data suggest brief exposure of

DCs to proliferating Nm was required to induce poor

maturation. It also implies that bacterial proliferation or

protein synthesis is required for the effect of the livebacteria.

Distinct DC maturation phenotype response to live

meningococci is dominant over the stimulus of killed

bacteria

In natural infection it is likely there is a combination of

viable and dead bacteria, so we investigated whether the

presence of both live and killed Nm affected the distinct

DC phenotype we observed from live Nm-stimulated DCs.

To test this, DCs were co-cultured with live and killed Nm

and the surface phenotype of DCs assessed as before by

flow cytometry. In the presence of live bacteria, fixed

bacteria were unable to induce DC maturation to levels

observed when incubated with fixed bacteria alone, as

illustrated in Fig. 4 for CD40, CD83, CD86 and HLA-Class

1 expression. The same pattern of results was observed

when the ratio of live to killed bacteria was varied (moi 100

killed and moi 10 live) or when LPS (a potent inducer of

DC surface maturation) was substituted for the killed

bacteria. These data show that distinct DC maturation in

response to live bacteria is dominant over normally potent

inducers of DC maturation.

Distinct DC surface phenotype in response to live

meningococci is dependent on contact between bacteria

and DC

We next investigated whether the pattern of surface

markers obtained on co-culture with live bacteria required

contact with DCs. Responses of DC to live and killed

meningococci, either in direct contact or separated by

0.1 mm transwell (preventing passage of intact meningo-

cocci but allowing free passage of membrane fragments

including neisserial blebs and liquid phase molecules),

were examined. As expected, when live bacteria and DCs

were not separated by the transwell membrane, little or no

induction of cell surface markers was observed, as illus-

trated in Fig. 5A using HLA-Class I expression as an

example. In contrast, when live bacteria were separated

by the 0.1 mm membrane, HLA-Class I was upregulated

(Fig. 5A). These results suggest that bacterial compo-nents released into the media are capable of inducing DC

maturation. To confirm these findings, DCs were incu-

bated with supernatants from live bacteria grown in mid-

log phase. We found that bacterial supernatant from live

bacteria was a potent stimulator of DC maturation

(Fig. 5B). Similar patterns were observed for CD83,

CD86, CD40 and HLA-DR (data not shown). We postulate

that a likely reason for this is the activity of inflammatory

moieties such as outer membrane vesicles (OMVs) or

released neisserial products (DNA and peptidoglycan),

which would be able to pass through the transwell mem-

brane or would be present in supernatant following growthof Nm. This is perhaps not surprising as it is known that

meningococcal OMVs are potent inducers of DC matura-

tion (Al Bader et al., 2003).

To further explore the mechanisms that may explain the

distinct DC phenotype observed in response to intact live

Nm, DCs were incubated with lysates from live bacteria.

As Fig. 6 demonstrates, lysates from live bacteria induced

upregulation of DC maturation markers (using CD40 and

HLA-DR expression as an example). Similar results were

also observed for killed bacterial lysates (data not shown).

In contrast, intact live Nm failed to induce CD40 expres-

sion and only moderate increase in HLA-DR expression

(Fig. 6 and as previously demonstrated).

Taken together, these data strongly suggest that the lack

of DC maturation induced by Nm is due to a mechanism

that requires close contact with bacteria and DC. The

finding that lysates of live bacteria induce similar matura-

tion to that seen with whole killed bacteria suggests that it

is not simply due to increase in production of a bacterial

product but rather a function of intact viable bacteria requir-

ing bacterial cell division or protein synthesis or both.

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Fig. 4. Distinct DC phenotypic response to live Nm is dominantover the response to killed bacteria. DCs were co-cultured with live,killed and live/killed Nm at moi 100 for 18 h. Expression of CD40,CD83, CD86 and HLA-Class 1 were then determined by flowcytometry. Flow cytometry profiles are from one donor out of fivestudied, with similar results.

2860 H. E. Jones et al.



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Live Nm induces different profiles of DC IL-12 p70 and

IL-10 production compared with killed bacteria

Our previous work has demonstrated that DC cytokine

responses to Nm, especially IL-12 p70 and IL-10, are

dependent upon internalization of bacteria by DC (Uronen-

Hansson et al., 2004a). Given the lack of induction of

co-stimulatory molecule expression in response to live

bacteria shown in this study, we wanted to determine

whether this would be reflected in poor cytokine responses

compared with killed Nm. Interestingly, we found that live

bacteria induced similar levels of IL-6 and TNF-a to those

we had previously published with killed Nm (Uronen-

Hansson et al., 2004a). However, when IL-12 p70 and

IL-10 production was examined, an entirely different pat-

tern emerged. As Fig. 7 illustrates, although IL-10 and

IL-12 production can be detected in response to both live

and killed meningococci, live Nm induced significantly

greater levels of IL-12 than killed Nm. This was significant,

despite interindividual variation of cytokine responses

to these bacteria (moi of 100 and 10, P= 0.007 and

P= 0.046, respectively, n= 10). In contrast, IL-10 produc-tion in response to killed organisms was significantly

greater than that seen with live bacteria (moi 100,

P= 0.001; moi 10, P= 0.005).

Internalization of live meningococci by human DC is

reduced compared with killed meningococci

Our previous work showed that phagocytosis of killed

Nm is critical for IL-12 production (Uronen-Hansson

Fig. 5. Distinct DC phenotype response tolive Nm requires contact between bacteriaand DC. DCs cultured in the lower chamberof a transwell were separated from thebacteria (moi 100) added to the upperchamber by a membrane with 0.1 mm pores.This was compared with DC responses in theabsence of transwell, but otherwise identicalculture conditions (A). In parallel with thisexperiment DCs were stimulated with the

supernatant equivalent to that released frommoi 100 of live and killed Nm (B). After 18 hexpression of HLA-Class I on DCs wasdetermined by flow cytometry. Data arerepresentative of three experiments from

three different donors yielding similar results.

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Fig. 6. Live Nm lysates are potent stimulators of DC maturation.Nm lysates were prepared with 0.05% NP40 detergent(Experimental procedures). DCs were stimulated with live Nmlysate equivalent to an moi of 10. After 18 h expression of CD40and HLA-DR was compared with levels induced by live and killedNm (moi 10) by flow cytometry. NP40 detergent alone had no effecton DC maturation (data not shown).




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protein (eGFP). The difference in percentage of DCs

associated with bacteria were clearly reduced in live DC

co-culture compared with those exposed to killed bacteria

(Fig. 8B). These data further confirm that results with the

FITC-labelled bacteria are valid for the time period (06 h)

studied.

We confirmed our flow cytometric analysis of phagocy-

tosis by using confocal laser scanning microscopy

(Uronen-Hansson et al., 2004a). Figure 9 shows DC incu-

bated with killed (Fig. 9A) and live (Fig. 9B) bacteria after

4 h of incubation. Internalized bacteria were assessed by

examining optical sections using ALEXA 586-conjugated

HLA-DR antibody as a counterstain. To distinguish

between internalized and surface-bound bacteria, a differ-

ential antibody staining technique with antibody specific for

porin A serosubtype 1.7 was used to detect if bacteria were

bound at the surface. In these experiments, surface-bound

bacteria were FITC positive and stained with ALEXA 568-

conjugated porin antibody. They appeared as red or yellow

if overlay was used whereas internalized bacteria appear

green only, as the cells were not permeabilized and the

bacteria were inaccessible to antibody binding.

IL-12 p70 and IL-10 production by DC in response to

live and killed Nm are dependent upon phagocytosis

The results presented in the previous section initially ap-

peared at odds with our previous work (Uronen-Hansson

Fig. 9. Nm internalization by DCs. DCs wereincubated with either killed (A) or live (B)FITC-labelled Nm for 1, 2, 4 and 6 h (4 h timepoint shown here). Cells were allowed toadhere to adhesion slides and then stainedwith either anti-HLA-DR antibody (i), orTo-Pro3 to stain the nucleus (ii), or acombination of To-Pro3 andanti-meningococcal serosubtype P1.7antibody followed by Alexa Fluor 568 goat

anti-mouse IgG (H+L) (iii) and then visualizedby scanning laser confocal microscopy.




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et al., 2004a) as we would expect that high IL-12 produc-

tion would be associated with efficient phagocytosis of

meningococci. Our previous studies with killed bacteria

showed that IL-12 production could be abrogated when

experiments were performed in the presence of cytocha-

lasin D, a potent inhibitor of phagocytosis. One possibility

was that in contrast to what occurs with killed bacteria,

IL-12 production in response to live bacteria may not be

dependent on internalization. However, as Fig. 10 clearly

demonstrates, blocking internalization by adding cytocha-

lasin D prior to co-culture with live bacteria and DCs

completely abrogated IL-12 p70 production. In addition, we

also found complete inhibition of IL-10 production in

response to both live and killed Nm by cytochalasin D.

Interestingly, cytochalasin D has not effect on IL-6 and

TNF-a production and DC maturation profiles for menin-

gococcal stimulated DCs (Uronen-Hansson et al., 2004a).

These data show that internalization of live bacteria is

required for IL-12 and IL-10 production and confirm that

phagocytosis of Nm is required for production of these two

important cytokines but is not essential for DC maturation.

Blocking exogenous IL-10 activity restores IL-12 p70

production by DCs to killed Nm

The reason behind the disparity between IL-12 and IL-10

production in DCs in response to live and killed meningo-

cocci required clarification. Given the dependence of

phagocytosis for the generation of both of these cytokines

to both killed and live bacteria, we postulated that IL-12

production may be reduced by activity of exogenouslyreleased IL-10. To test this possibility, DCs were pre-

incubated with a blocking antibody to IL-10 prior to

co-culture with DCs and killed bacteria. Levels of detect-

able IL-10 in supernatants from stimulated DCs were

reduced to near baseline levels (Fig. 11). Blocking the

activity of endogenously released IL-10 was found to

restore IL-12 production by DCs in response to both killed

and live bacteria (Fig. 11). No effect was seen when a

control antibody was added. We conclude that the differ-

ential production of IL-12 in response to live and killed

bacteria may be to a large part controlled by the action of

released IL-10. As IL-10 production in response to killed

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Fig. 10. Blocking phagocytosis inhibits production of IL-12 p70 andIL-10 by human DC in response to both live and killed Nm. DCswere incubated with live or killed Nm (moi 100) with or without10 mg ml-1 cytochalasin D (CD). After 18 h supernatants wereharvested and analysed for IL-10 and IL-12 by ELISA. Results areexpressed as the mean and standard error of the mean of fourseparate experiments.

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bacteria is higher than in response to live bacteria, the

effect seen on IL-12 production stimulated by killed Nm is

greater.

Discussion

Dendritic cells provide a critical link between the innate and

adaptive immune response. What has emerged in recent

years is the requirement for coordinated antigen presenta-

tion, together with signals via the action of cytokines and

chemokines and receptor cross-talk between APC and

T-cells to achieve optimal adaptive immune responses

(Banchereau et al., 2000). It is increasingly clear that

engagement of microbial products such as LPS, with host

pathogen recognition systems, predominantly TLRs, dic-

tates these signals (Kapsenberg, 2003; Pulendran and

Ahmed, 2006). There is evidence that perturbations in

these signals can have important and lasting conse-

quences for the nature of adaptive immune responses to

microbes and vaccine components derived from them. It is

noteworthy that certain vaccine preparations that havesuboptimal immunogenicity in vivoalso lack the capacity to

stimulate DCs in vitro (Skowera et al., 2005). In contrast,

the success of some vaccines appears to be as a result of

their capacity to engage multiple TLR ligands on DCs

(Querec et al., 2006). Further understanding on how Nm

interacts with DCs in terms of induction of co-stimulation

molecules and cytokines provides a logical strategy for

improving the efficacy of vaccines against Nm.

Our previous work has shown that killed Nm are an

effective way to target DCs because they are readily

phagocytosed. We have also shown that expression of

LPS on Nm is essential for uptake and subsequent cytok-ine production by DCs (Uronen-Hansson et al., 2004a).

This current study adds an extra dimension to the story as

it shows that, even taking into account the presence of

adjuvant properties of meningococcal LPS, viable dividing

bacteria behave differently in their capacity to stimulate

DCs compared with killed bacteria and even viable bac-

teria that are not dividing. Despite the fact that live men-

ingococci are able to induce cytokine production, they

appear remarkably inefficient at inducing increased

expression of DC maturation markers. To our knowledge,

this has not been demonstrated before. These data raise

the possibility that Nm may have the capacity to interfere

with or modulate DC function. This is supported by data

presented in this study that show the phenotypic response

to live meningococci is dominant over the normally potent

stimulus of maturation.

The finding that Nm can modulate DC function is not

without president in other organisms. For example, a

number of pathogens appear to be able to target and

interfere with DC function resulting in ineffective cellular

and humoral immunity. Microbial pathogens achieve this

via a number of different mechanisms including direct

cytotoxicity or specific interference of DC signalling path-

ways required for effective cytokine responses or matu-

ration (Urban et al., 1999; Agrawal et al., 2003; Skinner

et al., 2004; Tobar et al., 2004; Marketon et al., 2005).

It is not known whether the lack of upregulation of cell

surface markers in DCs in response to live Nm shown in

this study is likely to have any effect on subsequent T-cell

responses. Co-stimulatory molecules are still expressed

on the DCs exposed to live bacteria, albeit at a lower level

than seen in response to killed organisms, and this may

still allow for efficient interactions of DC with T-cell.

However, there is evidence from other work that it is not

only the expression of these molecules (e.g. CD40 and

CD86) but also the density of these receptors that can

have important effects upon DCT-cell interactions.

In addition to the lack of co-stimulatory molecule

induction, there was only modest or no expression of

HLA-Class I and HLA-DR on DCs stimulated by live bac-

teria. While the significance of this remains to be eluci-

dated, this could influence the nature of the DCT-cellsynapse, as antigen density is a determinant of T-cell

polarization (Langenkamp et al., 2000). Further work in

our laboratory is investigating whether efficiency of

antigen presentation is also reduced in DC stimulated

with live meningococci.

One important matter raised by these data is: what are

the mechanisms that may be operating that result in the

poor maturation response? We have shown that viable but

non-proliferating (cm treated) Nm induce a DC phenotype

similar to that induced by killed Nm. As cm rapidly blocks

bacterial protein synthesis, there is a possibility that men-

ingococci, upon contact with DC, increase expression offactors that may directly interfere with processes leading to

increase in expression of maturation markers on DC.

However, the finding that lysates of live bacteria are

stimulatory to DCs does not support the notion that

over-production of bacterial products alone is responsible

for the lack of maturation seen with live bacteria.

As the effect of live bacteria requires contact and is

dominant over normally potent maturation signal such as

killed bacteria or LPS, we propose that the likely mecha-

nism is an active process involving a functional surface

expressed component of meningococci. We cannot rule

out the activity of molecules locally released by either

bacteria or DCs in response to live infection. However,

as neither supernatants from bacteria alone (seen in

Fig. 5 in this study) nor indeed supernatants from DC

live bacterial co-cultures (H. Jones, pers. comm.) display

this inhibitory/dominant effect on DC maturation we feel

this is unlikely. There are a number of potential menin-

gococcal candidate genes that could potentially affect

host cell function. Work is in progress to identify genes

and factors that might be suppressing phenotypic




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expression of DC co-stimulatory molecules and HLA-

Class I and HLA-DR. Clearly, the identity of such factors

would be advantageous as these could potentially be

deleted or modified in attenuated strains that could be

used in novel vaccines preparations.

This study focused on the DC response to one strain of

wild-type capsulated bacteria (H44/76). However, work

carried out by two of the authors (G.L.J.D., H.E.J.)

showed that serogroup B strains B1940 and MC58 have

identical disparity of DC maturation responses between

live and killed organisms. Furthermore, we have not found

any influence of capsulation or modification of LPS struc-

ture on the distinct maturation phenotype of DCs, as

mutants lacking these components behave the same way

as wild-type strains (G.L.J. Dixon, unpubl. data).

The other clear difference between live and killed bac-

teria shown in these studies is the difference in rate of

their internalization by human DC. Other workers have

shown that live Nm are internalized by DC, and this is also

affected by capsulation status and LPS oligosaccharide

structure (Kurzai et al., 2005). Most of these studies wereperformed in serum-low or even serum-free conditions,

and therefore differ to our studies which do include serum.

We have previously shown that in the absence of serum

factors, uptake (presumably by non-opsonic mecha-

nisms) does occur at a low level but this greatly enhanced

by both serum and specifically by lipopolysaccharide-

binding protein (LBP) (Uronen-Hansson et al., 2004a).

Accordingly, one likely hypothesis is that differences in

uptake between live and killed organisms are due to inef-

ficiency of receptor engagement. It is also likely that this is

dependent on the presence of serum factors such as LBP,

soluble CD14 and RGD motif containing factors such asvitronectin and fibronectin. At present it is unclear which

receptors are critical for uptake of Nm by DC, but under

non-opsonic conditions there is indirect evidence that

scavenger receptors support internalization of unencap-

sulated Nm into DC (Kurzai et al., 2005). Recent work we

have performed has suggested that b2-integrins, involving

LBP as an opsonin, may facilitate uptake of killed Nm (our

manuscript in preparation). It would be interesting to see

whether this pathway operates efficiently with respect to

live bacteria uptake. The differences between rate and

efficiency of uptake of killed Nm compared with live and

the difference in cytokine responses may be linked. We

show that both IL-10 and IL-12 production is dependent

upon bacterial uptake by DC. However, we also show that

that IL-10 release by DC reduces IL-12 production, and

this may help to explain the unexpected finding that killed

bacteria, which are phagocytosed more efficiently than

live, produce less IL-12. While increase in effective dose

of live bacteria is a potential confounding factor in these

experiments, this cannot explain the increase in potency

of live bacteria for inducing IL-12 production in DCs, as

uptake of live bacteria is consistently far lower than that of

killed bacteria even in the presence of increased bacteria

division in live cultures.

It is not known what are the consequences of the dif-

ferent profiles of IL-10 and IL-12 p70 induced by live and

killed meningococci on T-cell responses. It is generally

accepted that Gram-negative bacteria such as Nm are

potent inducers of T-helper type 1 responses (de Jong

et al., 2002) and the likely reason for this is the propensity

of LPS in these bacteria to induce production of the IL-12

family of cytokines by DCs (Trinchieri, 1993; Pulendran

et al., 2001). Production of IL-12 by DCs is responsible for

multiple effects on lymphocytes, in particular promoting

IFN-g production by CD4 cells, favouring B-cell differen-

tiation and isotype switching to Th1-associated immuno-

globulin IgG2, considered to be most effective isotype

against polysaccharide antigens (Trinchieri, 1998). In con-

trast, IL-10 released by DCs exerts other effects, such as

the class switching of IgD bearing B-cells to produce IgG1

and IgG3 antibody (Briere et al., 1994). This is pertinent

as bactericidal antibodies against the subcapsular proteinantigens such as porA, induced by both OMV vaccines

and after natural infection, are of IgG1 and IgG3 isotypes

(Sjursen et al., 1990; Naess et al., 1999; Martin et al.,

2001). Although IL-10 is considered an anti-inflammatory

cytokine (Igietseme et al., 2000) it may have effects that

could influence the T-cell priming capacity of meningococ-

cal pulsed DCs. For example, IL-10 promotes DC survival,

and this may be important for maintaining contact

between DC and Ag-specific T-cell.

In this article, we present data that show live meningo-

cocci induce different pattern of maturation/activation

from that seen with dead bacteria. The biological conse-quences of these different DC phenotypes will become

apparent when tested in a system that assesses the

effects on meningococcal stimulated DCs on antigen-

specific T-cell responses and subsequent effects on B-cell

function. Our findings may be important for meningococ-

cal vaccine development.

Experimental procedures

Bacteria

The Nm strain used in this study H44/76 (B:15:P1.7,16:) is

encapsulated and piliated and expresses L3 immunotype (Holten

et al., 1979). Bacteria were grown on GC agar base (BD Bio-

sciences, Oxford, UK) supplemented with 1% Vitox (Oxoid) and

cultured in 5% CO2 in air at 36C. Bacteria were grown overnight

(18 h) and then suspended in RPMI 1640 medium without phenol

red (Invitrogen). Optical density was measured at 540 nm and a

suspension at OD 1.0 was determined to contain 1 109 bacte-

ria ml-1 by serial dilution and plating using standard microbiologi-

cal techniques. Bacteria were fixed in 0.5% paraformaldehyde for

20 min and then washed extensively. For fluorescent labelling,

live Nm were incubated with 0.5 mg ml-1 FITC (Sigma, Poole,




12/14

UK) for 20 min at 37C and then an aliquot was taken and then

killed as described above. The level of FITC incorporation was

confirmed by flow cytometry. In previous optimization experi-

ments, method of killing bacteria (UV irradiation, heat treatment

for 56C for 30 min) does not alter the pattern of DC activation or

cytokine production (H. Uronen-Hansson, pers. comm.). To gen-

erate bacteria expressing green fluorescent protein, wild-type

bacteria were transfected with plasmid containing enhanced

green fluorescence protein under PorA promoter (Christodoul-

ides et al., 2000). This plasmid was modified by replacement of

ampicillin resistance cassette with ermC gene conferring resis-

tance to erythromycin (kind gift from Peter van der Lay, NVI,

Bilthoven, the Netherlands). Successful transfectants were

screened for fluorescence and subcultured onto GC agar plates

containing 50 mg ml-1 erythromycin. Level of fluorescence was

checked by flow cytometry and immunofluorescence microscopy.

For studies using cm (clinical grade obtained from Pharmacy,

Great Ormond Street Hospital, UK), optimization of time kill

curves was performed in our tissue culture system. At doses of

2.510 mg ml-1, and at concentrations over 105-107 colony-

forming units (cfu) ml-1, bacterial concentration remained static

over incubation period of up to 18 h, as determined by serial

dilution and plating and quantifying bacteria after further over-

night incubation on GC agar plates.

For preparation of bacterial lysates 2 108 cfu ml-1 were sus-

pended in 0.05% NP40 detergent (Sigma) in PBS and mixed

rigorously for 15 min. Cellular debris was removed by centrifuga-

tion and 0.2 mm filtration. Lysis of live bacteria was confirmed by

plating lysates on GC agar overnight.

Dendritic cell culture and activation

Dendritic cells were generated from human peripheral blood

mononuclear cells (PBMCs) as described previously (Sallusto

and Lanzavecchia, 1994; Dixon et al., 2001) but with some

modifications. Briefly, monocytes were prepared from PMBCs

using positive selection using CD14 immunomagnetic beads

(Miltenyi Biotec, Surrey, UK). CD14-isolated cells were then cul-

tured in RPMI supplemented with 10% FCS, 2.4 mM L-glutamine

(all from Invitrogen), 100 ng ml-1 human recombinant GM-CSF

and 50 ng ml-1 human recombinant IL-4 (obtained from Professor

Benjamin Chain). DCs were used after 57 days of culture and

phenotype was determined by flow cytometry (FACS calibur BD

Biosciences, Oxon, UK). Immature DCs were CD3-negative,

CD14-low, CD19-negative, CD83-negative, CD25-negative and

expressed low levels of HLA-DR, HLA-DQ, HLA-Class 1, CD40,

CD86 and CD1 as previously described (Dixon et al., 2001).

For stimulation experiments, DCs (5 105 ml-1) were cultured

with live or killed Nm at an moi of 100, 10 or 1 for 1824 h. In

some experiments, 2.510 mg ml-1 cm was added to the DC

bacteria co-culture either at the start of stimulation or at various

time points after addition of bacteria, medium or LPS. Prior to

staining, an aliquot of DC culture was stained with trypan blue

(Sigma Aldrich, Poole, UK) to assess the proportion of cell death

in co-cultures using a haemocytometer.

For transwell experiments, DCs were added to 24-well tissue

culture plates at a density of 5 105 cells ml-1. The 0.1 mm pore

size inserts were placed in contact with media and the bacteria

were added to the top chamber. After overnight culture, DCs were

removed and cell surface markers analysed as described below.

Lack of any ingress of live meningococci to the lower chamber

was proven by culturing an aliquot of the stimulated cells on GC

agar.

Surface marker expression and cytokine measurement

Expression of surface molecules associated with DC maturation

and activation (CD40, CD83, CD86, HLA-Class 1 and HLA-DR)

was detected by staining with 2 mg ml-1 of the appropriate FITC-or phycoerythrin (PE)-conjugated monoclonal antibodies (all

Caltag Medsystems, Silverstone, UK) for 30 min on ice. Follow-

ing incubation, cells were washed with phosphate-buffered saline

(PBS) with 0.2% bovine serum albumin (BSA) and then fixed in

1% formaldehyde. Cells were then analysed by flow cytometry on

a FACScalibur using CellQuest Pro software (BD Biosciences

Oxford, UK). DCs form a discrete population when separated by

side and forward scatter parameters; this population formed the

collection gate and at least 5000 events within this gate were

collected for analysis.

Cytokine (IL-6, IL-1b, TNF-a, IL-12 p70 and IL-10) production

in cell culture supernatants of co-culture experiments was carried

out by eBioscience ELISA kits (Insight) following the manufactur-

ers instructions. Lower limit of detection of IL-12 and IL-10 are4 pg ml-1 and 2 pg ml-1 respectively.

Phagocytosis assay

The association of Nm with DCs was determined by flow cytom-

etry and confocal microscopy. DCs were incubated with FITC-

labelled Nm and then aliquots of cells taken at various time

points, as previously described (Uronen-Hansson et al., 2004a).

Cells were separated into two aliquots. One was washed exten-

sively with PBS and 0.2% BSA, fixed with 1% formaldehyde then

analysed by flow cytometry. DC and Nm association was shown

by the expression FITC within the DC-gated population. To facili-

tate gating, DCs were counterstained with phycoerythrin-conjugated DC-SIGN antibody, which is expressed highly on DCs

regardless of maturation status. In some experiments, DC asso-

ciation with meningococci was judged by per cent positive events

and mean fluorescence intensity in DC-SIGNbright events. Using

this method, a clear distinction between DC associated and non-

associated with bacteria could be achieved. Inhibition of phago-

cytosis was achieved using the inhibitor of actin polymerization

cytochalasin D (Sigma) at 10 mg ml-1, which was added to DCs

just prior to incubation with Nm. In optimization experiments,

addition of cytochalasin D prior to co-culture with fluorescent

bacteria reduced bacteria fluorescence to near background

levels, showing that the majority of FITC/GFP-positive events are

due to bacteria that have been internalized by DC.

The remaining aliquot of cells were washed with PBS and then

allowed to adhere to poly lysine-coated slides for 10 min (Paul

Marienfeld Gmbh and CO, Germany). Cells were then fixed with

4% paraformaldehyde. In some experiments, DCs were visual-

ized by either staining the nuclei with To-Pro3 (Molecular Probes,

Cambridge Biosciences, Cambridge, UK) or staining the DC

surface with 5 mg ml-1 anti-HLA-DR antibody (Dako, Glostrup,

Denmark) followed by Alexa Fluor 568 goat anti-mouse IgG

(Molecular Probes). To determine if Nm was internalized or

surface bound, extracellular bacteria were identified by staining

with 10 mg ml-1 P1.7 antibodies specific for Nm (NIBSC, South




13/14

Mimms, UK) followed by Alexa Fluor 568 goat anti-mouse IgG

(Invitrogen Molecular Probes, Paisley, UK). Slides were then

washed and mounted with Vectashield (Vector laboratories,

Peterborough, UK) and then images were obtained using a Leica

SP2 confocal laser scanning microscope system (Leica, Milton

Keynes, UK). To identify internalized bacteria at least 10 optical

sections (0.20.5 mm) spanning the entire DC were visualized by

Leica confocal imaging software.

IL-10 inhibition studies

Prior to incubation with bacteria, DCs were incubated with

30 mg ml-1 anti-IL-10 blocking antibody (R&D Systems, Oxon,

UK) for 10 min.

Viability

The percentage of viable cells was assessed by trypan blue

staining of an aliquot of cells prior to fixation using a

haemocytometer. In all culture conditions, a proportion of cells

(ranging from 5% to 15%) were trypan blue positive. However,

there was no difference observed in the proportion in culturesstimulated with medium, live or killed Nm. The proportion of DCs

undergoing apoptosis was measured by the detection of active

caspases using a Vybrant FAM Poly Caspase Assay Kit

(Molecular probes) according to manufacturers instructions.

Statistical analysis

Where shown, statistical analysis was performed using SPSS

version 12.01 software. Due to variability of biological responses

(such as individual variation of cytokine responses to microbial

derived products and the arbitrary unit of fluorescent intensities

derived from flow cytometric analysis) paired t-tests were per-

formed on individual donor-derived DC responses to live and

killed bacteria. The number of donors studied allowed for para-

metric testing to be performed.

Acknowledgement

This work was supported by Meningitis UK.

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