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Microbes and Infection xx (2014) 1e9 www.elsevier.com/locate/micinf

Original article

Human dendritic cell maturation and cytokine secretion upon stimulation with Bordetella pertussis filamentous haemagglutinin Q7

Violette Dirix a,b,c,d,1, Nathalie Mielcarek a,b,c,d, Anne-Sophie Debrie a,b,c,d, Eve Willery a,b,c,d, Sylvie Alonso a,b,c,d,2, Virginie Versheure e, Franc¸oise Mascart e,f, Camille Locht a,b,c,d,* a

INSERM U 1019, Lille, France CNRS, UMR8204, Lille, France c Univ Lille Nord de France, Lille, France d Institut Pasteur de Lille, Center for Infection and Immunity of Lille, Lille, France e Laboratory of Vaccinology and Mucosal Immunity, Universite´ Libre de Bruxelles (U.L.B.), 808 route de Lennik, 1070 Brussels, Belgium f Immunobiology Clinic, Hoˆpital Erasme, Universite´ Libre de Bruxelles (U.L.B.), 808 route de Lennik, 1070 Brussels, Belgium b

Received 6 March 2014; accepted 22 April 2014

Abstract In addition to antibodies, Th1-type T cell responses are also important for long-lasting protection against pertussis. However, upon immunization with the current acellular vaccines, many children fail to induce Th1-type responses, potentially due to immunomodulatory effects of some vaccine antigens, such as filamentous haemagglutinin (FHA). We therefore analysed the ability of FHA to modulate immune functions of human monocyte-derived dendritic cells (MDDC). FHA was purified from pertussis toxin (PTX)-deficient or from PTX- and adenylate cyclasedeficient Bordetella pertussis strains, and residual endotoxin was neutralized with polymyxin B. FHA from both strains induced phenotypic maturation of human MDDC and cytokine secretion (IL-10, IL-12p40, IL-12p70, IL-23 and IL-6). To identify the FHA domains responsible for MDDC immunomodulation, MDDC were stimulated with FHA containing a Gly/Ala substitution at its RGD site (FHA-RAD) or with an 80kDa N-terminal moiety of FHA (Fha44), containing its heparin-binding site. Whereas FHA-RAD induced maturation and cytokine production comparable to those of FHA, Fha44 did not induce IL-10 production, but maturated MDDC at least partially. Nevertheless, Fha44 induced the secretion of IL-12p40, IL-12p70, IL-23 and IL-6 by MDDC, albeit at lower levels than FHA. Thus, FHA can modulate MDDC responses in multiple ways, and IL-10 induction can be dissociated from the induction of other cytokines. Ó 2014 Published by Elsevier Masson SAS on behalf of Institut Pasteur.

Keywords: Bordetella; Filamentous haemagglutinin; Human dendritic cells

1. Introduction

* Corresponding author. Center for Infection and Immunity of Lille, Institut Pasteur de Lille, 1, rue du Prof. Calmette, F-59019 Lille Cedex, France. Tel.: þ33 3 20 87 11 51; fax: þ33 3 20 87 11 58. E-mail addresses: [email protected], [email protected] (C. Locht). 1 Current address: Laboratory of Vaccinology and Mucosal Immunity, Universite´ Libre de Bruxelles (U.L.B.), 808 route de Lennik, 1070 Brussels, Belgium. 2 Current address: National University of Singapore, Centre for Life Science #03-05, 28 Medical Drive, 117597 Singapore, Singapore.

Pertussis remains one of the most poorly controlled vaccine-preventable childhood diseases in the world, with still 200,000e300,000 estimated deaths each year [1], even though the whooping cough incidence has dramatically decreased [2] following the introduction of systematic vaccination with the first-generation whole-cell pertussis vaccines since the 1940s. Since the end of the 20th century, whole-cell vaccines were progressively replaced, at least in industrialized countries, by new generation, acellular vaccines [3]. Unexpectedly, pertussis is on the rise again, with epidemics even in countries with high

http://dx.doi.org/10.1016/j.micinf.2014.04.003 1286-4579/Ó 2014 Published by Elsevier Masson SAS on behalf of Institut Pasteur. Please cite this article in press as: Dirix V, et al., Human dendritic cell maturation and cytokine secretion upon stimulation with Bordetella pertussis filamentous haemagglutinin, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.04.003

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vaccination coverage, such as the US, resulting in more cases in 2012 than ever observed since 1955 [2]. Several recent studies have shown that protection induced by the acellular vaccines wanes significantly faster than that induced by whole-cell vaccines [4e6], stressing the need for a better understanding of vaccine-induced immunity against pertussis. In addition to antibodies known for long to participate in protection against Bordetella pertussis infection, the role of specific cellular immune responses is also now well recognized [7]. Children with whooping cough, both in the acute phase and during convalescence, induce a Th1-type immune response [8,9]. Similarly, whole-cell vaccines induce specific interferon-gamma (IFN-g) responses, whereas the acellular vaccines typically induce a mixture of Th1- and Th2-type cytokines [10e12]. We have previously reported that the monocytes of children with poor IFN-g responses upon vaccination with acellular pertussis vaccines spontaneously secreted high levels of interleukin (IL)-10, and identified IL-10 as an important limiting factor for the development of specific Th1-type responses to B. pertussis antigens [13]. Filamentous haemagglutinin (FHA) is one of the main antigens present in virtually all pertussis vaccines and was reported to supress the LPS-induced IL-12 production by alveolar macrophages through an IL-10-dependent mechanism in mice [14]. Tr1 responses specific for FHA were also reported in mice as a potential strategy for B. pertussis to escape from protective Th1 responses [15]. Virus-specific IFN-g production in lymph nodes from influenza virus-infected mice is also suppressed by FHA [16], as is the development of IL17-mediated inflammatory responses [17] in mice. Therefore, FHA is a good candidate as an inducer of IL-10 upon immunization with acellular pertussis vaccines. However, it has not yet been established whether FHA exerts these immunemodulatory effects on human antigen-presenting cells, and it is not yet known whether such immune-modulatory functions can be physically separated from antigenic properties of FHA. Here, we therefore tested different molecular forms of FHA for their ability to induce phenotypic maturation of and cytokine-secretion by carefully characterized human monocyte-derived dendritic cells (MDDC), in order to (i) establish whether FHA has immune-modulatory functions on human MDDC and (ii) whether the putative immunemodulatory properties of FHA can be separated from its antigenic properties.

Table 1 B. pertussis strains used in this study. B. pertussis strain BPRA BPSA207 BPSA201 BPSA87

Relevant phenotype þ



FHA PTX FHAþ PTX ACT FHA(G1098A)þ PTX Fha44þ PTX

Reference [23] This study This study [34]

medium containing 1 g/l of heptakis (2,6-di-o-methyl) bcyclodextrin (Sigma Chemical Co.). B. pertussis BPSA201 was obtained by allelic exchange as described by Stibitz [20], using pAS88 to integrate the Gly1098Ala point mutation into the fhaB gene in the chromosome of PTX-deficient B. pertussis BPRA. Consequently, this strain expresses the mutated FHA-RAD protein instead of FHA. pAS88 resulted from the insertion of the 709-bp BamHI fragment from pAS87 into BamHI-linearized pAS70. pAS87 is a pUC18-derivative in which the BamHI-digested PCR1 product was inserted, and pAS70 is a pJQmp200rpsL18derivative [21] digested with BamHI and SalI and in which the BamHIeSalI-digested PCR2 product was inserted. PCR1 and PCR2 were obtained by polymerase chain reaction (PCR) on BPRA chromosomal DNA and using oligonucleotides 50 GACGTGCGCGGCGGATCCACG-30 and 50 -ATGCG0 GATCCGCGCGCCCGAC-3 and the oligonucleotides 50 AGCGTCGGGCGCGCGGATCC-30 and 50 -CCAGCTTGCCACGTCGACGCT-30 , respectively. The point mutation leading to the Gly-1098 to Ala change was generated by the modification of the GC nucleotides to CG (underlined). The amplicon encompassed nucleotides 2824e3562 (for PCR1) and 3532e4434 (for PCR2) of the fhaB gene. To construct B. pertussis BPSA207, A 545-bp fragment was amplified by PCR from the BPRA chromosome using oligonucleotides 50 -AAGCTTGACCGGGAGTCTGG0 0 CATCC-3 and 5 -GGTACCCGATATCCGCCGTCAGTGG-30 as primers. The amplicon was then inserted into pCRII-TOPO (Invitrogen), and the recombinant plasmid was digested with HindIII and KpnI, and the resulting 545-bp and inserted into the integrative plasmid pFUS2 [22] digested with the same restriction enzymes. The recombinant plasmid was then introduced into B. pertussis BPRA as described [23], and the BPRA-derivatives containing the pFUS2 integration were selected by growth on BG blood agar containing gentamycin. The adenylate cyclase toxin (ACT)-deficient phenotype was verified by the loss of haemolysis on BG blood agar plates.

2. Materials and methods 2.2. Antigens and reagents 2.1. B. pertussis strains and growth conditions The B. pertussis strains used in this study are listed in Table 1. They all derived from the Tohama I strain [18] and were grown on BordeteGengou (BG) agar (Difco, Detroit, MI) supplemented with 1% glycerol, 20% defibrinated sheep blood and 100 mg/ml streptomycin and 10 mg/ml gentamycin when needed (Sigma Chemical Co., St. Louis, MO) at 37  C for 72 h. Liquid cultures of B. pertussis were performed as described previously [19] in modified StainereScholte (SS)

FHA was purified from B. pertussis BPRA, BPSA201 or BPSA207 and Fha44 from BPSA87 as described previously [24] (Table 1). In all strains the ptx genes were deleted to avoid any possible contamination with pertussis toxin (PTX) during purification by heparin-sepharose chromatography. In BPSA207 the cya genes were deleted in addition, to also avoid contamination with ACT. FHA-RAD contains a single amino acid substitution in RGD sequence (G1098A) of FHA. Fha44 is the 80-kDa N-terminal moiety of FHA.

Please cite this article in press as: Dirix V, et al., Human dendritic cell maturation and cytokine secretion upon stimulation with Bordetella pertussis filamentous haemagglutinin, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.04.003

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The antigen concentrations were estimated by the BCAÔ Protein Assay Reagent Kit (Pierce Biotechnology, Rockford, IL), and the concentration of residual endotoxin was measured by the LAL test (kit QCL-1000, Lonza, Verviers, Belgium). The different FHA and Fha44 forms were used at a final concentration of 1, 5 or 10 mg/ml. Escherichia coli lipopolysaccharide (LPS) was purchased from Sigma Chemical Co. and used at a concentration of 1 mg/ml as a control. Human rGM-CSF and rIL-4 were from R&D Systems (Abington, UK) and Brucells (Brussels, Belgium), respectively. Polymyxin B was purchased from Sigma Chemical Co. 2.3. Purification and culture of monocyte-derived dendritic cells (MDDC) Human peripheral blood mononuclear cells (PBMC) were purified as described [11] from peripheral blood of healthy blood donors (Red cross, Leuven, Belgium) and incubated at approximately 10  106/ml (20 ml/T75 flask, BD Biosciences, San Jose, CA) in RPMI 1640 medium (Lonza) supplemented with 10% heat-inactivated LPS-screened foetal calf serum (FCS, Hyclone, Logan, UT), 0.1 mM non-essential amino acids (Life technologies, Paisley, UK), 2 mM L-glutamine (Life technologies), 40 mg/ml geomycin (ScheringePlough, Heist-op-den-Berg, Belgium), and 0.05 mM 2mercaptoethanol (Life technologies) (hereafter defined as complete medium) at 37  C under 5% CO2. After 2 h incubation, non-adherent cells were removed, and residual cells were cultured in complete medium in the presence of rGMCSF (800 U/ml) and rIL-4 (200 U/ml). Two days later, rGM-CSF and rIL-4 were again added to the cultures at 800 U/ ml and 200 U/ml, respectively. After an additional three days, immature MDDC were washed and analysed by flow cytometry for the expression of surface markers as described below. The MDDC were selected for further experiments only when the cell preparations contained >90% MDDC based on morphology, low expression of CD40, CD80, CD83, CD86 and HLA-DR, and absence of CD14, as evidenced by flow cytometry. These well definite limits were not always reached, and therefore some MDDC preparations were not selected for further experiments. As we used buffy coats, which did not reach the classical criteria to be used for blood transfusion, they were generally 16e24 h old, and the MDDC preparation was therefore not always efficient. MDDC (0.5  106 cells/ml) were resuspended in complete medium and stimulated with the different forms of FHA or LPS in 48-well plates (BD Biosciences) at 37  C and 5% CO2 for 24 h. Indicated concentrations of polymyxin B were added to neutralize residual endotoxin. After 24 h, the MDDC were harvested for immunophenotypic analyses, and the supernatants were collected for cytokine measurements by ELISA. 2.4. Immunophenotypic analyses MDDC were washed and resuspended in FACS Flow (BD biosciences), incubated for 30 min at 4  C with the following fluorochrome-conjugated monoclonal antibodies (mAbs): anti-

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CD14, -CD40, -CD80, -CD83, -CD86 and -HLA-DR. All mAbs were obtained from BD Biosciences, except for antiCD83, which was purchased from Immunotech (Marseille, France). The cells were analysed on a FACSCANTO (BD Biosciences), and the data were analysed using the FlowJo software (Treestar, Ashland, OR). 2.5. Cytokine measurements Cell culture supernatants were collected after 24 h incubation as described above, and cytokine concentrations were measured by ELISA. IL-10, IL-12p40, IL-6 (Cytoset, Life technologies), IL-12p70 (Quantikine, R&D Systems) and IL23 (Ready-SET-Go!, eBiosciences, Vienna, Austria) production was assessed by ELISA according to the manufacturer’s instructions. Sensitivities were 25 pg/ml for IL-10, IL-12p40, IL-6 and IL-23, and 0.5 pg/ml for IL-12p70. The optical density obtained was measured at 450 nm, and concentrations were calculated with the KC4 software (BRS, Drogenbos, Belgium). 2.6. Statistical analysis The results were analysed by nonparametric paired Wilcoxon or unpaired Mann Whitney tests using GraphPad Prism, version 4.00 for Windows (GraphPad Software, San Diego, CA, www.graphpad.com). 3. Results 3.1. Optimal FHA stimulation and MDDC culture conditions To study the effect of FHA on human MDDC, we adopted very strict quality control criteria for the MDDC and used them only when (i) they maintained a persistent immature immune-phenotype after 24 h of culture without any added stimulant, (ii) 1 mg/ml E. coli LPS induced detectable IL-10 secretion, (iii) polymyxin B inhibited the LPS-induced IL-10 secretion and (iv) in the absence of other stimuli up to 170 U of polymyxin B failed to induce phenotypic modulation and IL-10 production and (v) 1 mg/ml E. coli LPS increased their expression of CD40, CD80, CD83, CD86 and HLA-DR but not of CD14. The median (25the75th percentiles) increases of MFI after treatment with E. coli LPS compared to untreated MDDC were 1.9 (1.7e2.2) for CD40, 4.3 (2.9e5.6) for CD80, 3.5 (3.1e4.2) for CD83, 9.2 (4.2e13.6) for CD86 and 3.1 (2.2e5.4) for HLA-DR. The percentages of CD83þ cells were always higher than 63.1% (25the75th percentiles ¼ 69.2e82.9) after E. coli LPS treatment. In contrast, the percentage of CD14þ cells was always very low with median (25the75th percentiles) of 3.1% (0.6e4.8). When FHA was tested at different concentrations (1, 5 and 10 mg/ml) and incubation times in human MDDC cultures, IL10 could readily be detected in the culture supernatants after 18 h of incubation. After 24 h of incubation, the IL-10 concentrations in the supernatants started to decrease (data not

Please cite this article in press as: Dirix V, et al., Human dendritic cell maturation and cytokine secretion upon stimulation with Bordetella pertussis filamentous haemagglutinin, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.04.003

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shown). In further experiments, stimulation of MDDC was thus performed during 24 h. However, as the different purified FHA preparations used in this study contained variable, albeit low levels of residual B. pertussis LPS, as measured by the LAL test, the observed IL-10 secretion by the stimulated MDDC may potentially be due to the residual LPS within the FHA preparations. The LPS contents varied between 1.70 and 8.48 Endotoxin Units (EU)/ mg FHA (Table 2). The capacity of polymyxin B to neutralize the LPS [25] was therefore evaluated in the in vitro culture conditions used in this study. By using LPS from E. coli as a positive control and IL-10 release as a readout of the LPS effect, we found in preliminary experiments that the addition of 20 U polymyxin B in the culture medium neutralized 1 EU of E. coli LPS (data not shown). Although B. pertussis LPS is less active than E. coli LPS, we added 20 U polymyxin B/EU LPS to the FHA-containing MDDC culture medium for all further experiments. As illustrated in Fig. 1 the IL-10 concentrations released by MDDC stimulated in vitro with three different concentrations of FHA were not significantly different in presence or absence of polymyxin B, although for one subject, stimulated with FHA containing the highest concentration of LPS (8.48 EU/mg FHA), the addition of polymyxin B strongly decreased IL-10 secretion, especially when high concentrations of FHA were used. In this case, much of the IL-10 secretion in the absence of polymyxin B may thus be due to residual LPS. All subsequent experiments were therefore performed in the presence of polymyxin B. The largest amount of polymyxin B added was 170 U for the stimulation with FHA at 10 mg/ml. As a control, 170 U of polymyxin B was then added without antigen to the MDDC, and this resulted in no detectable IL-10 secretion (data not shown). Since the IL-10 concentrations secreted after stimulation with 5 mg/ml of FHA in the presence of adequate concentrations of polymyxin B were similar to those found after stimulation with 10 mg/ml of FHA, but higher than with 1 mg/ml of FHA, we used 5 mg/ml FHA for all further experiments. 3.2. Cytokine secretion after MDDC stimulation by different forms of FHA Since FHA can synergize with ACT [26] and is closely associated with ACT at the surface of B. pertussis [27], we could not formally rule out an effect of ACT on the FHAmediated IL-10 secretion by MDDC. We therefore purified

Table 2 Endotoxin levels in antigen preparations used in this study. Antigen preparation

Antigen concentration (mg/ml)

Endotoxin level (EU/mg antigen)

FHA

65 107 170 170 200

1.7 8.48 2.5 1.75 2.75

FHA-ACT FHA-RAD Fha44

Batch 1 Batch 2

Fig. 1. Effect of polymyxin B on the induction of IL-10 secretion by MDDC upon stimulation with FHA. MDDC from 5 different blood donors were stimulated with 1, 5 or 10 mg/ml FHA in the absence or presence of polymyxin B. Open and black squares represent results obtained with two different FHA preparations, containing 8.48 and 1.7 EU of LPS/mg FHA, respectively. Values obtained in the absence or presence of polymyxin B for a same donor are connected. Horizontal lines represent medians. IL-10 production was measured by ELISA in MDDC culture supernatants harvested after 24 h. NS ¼ non-significant.

FHA from B. pertussis BPSA207, an ACT- and PTX-deficient strain and tested its ability to induce IL-10 secretion by MDDC. As depicted in Fig. 2a, FHA purified from BPSA207 stimulated IL-10 secretion by MDDC at similar levels as FHA purified from the PTX-deficient ACT-producing strain BPRA, although both induced less IL-10 secretion than E. coli LPS, indicating that the observed IL-10 secretion was not due to contaminating ACT. Since the RGD site of FHA has been reported to be involved in the interaction of FHA with complement receptor 3 (CR3) [28,29], the CD61/CD47 complex [30] and the very late antigen-5 (VLA-5) integrin on human monocytes [31], it was of interest to investigate whether this site would be responsible for the FHA-mediated IL-10 secretion by MDDC. We therefore stimulated the MDDC with an FHA derivative produced by PTX-deficient B. pertussis BPSA201, a strain in which the RGD site of FHA was changed to RAD. Stimulation of MDDC with this molecule resulted in IL-10 secretion at levels similar to those observed after stimulation with native FHA (Fig. 2a), indicating that the RGD site of FHA is not essential for the induction of IL-10 by human MDDC. In addition to the RGD site, FHA also contains a carbohydrate recognition domain (CRD) [32], a heparin-binding domain (HBD) [18] and the mature C-terminal domain (MCD) [33] that may interact with different receptors on antigen-presenting cells. We therefore investigated the ability of Fha44 to induce IL-10 secretion by MDDC. This molecule represents the 80-kDa N-terminal moiety of FHA, lacking the RGD site, the CRD and the MCD, but containing its HBD. Fha44 purified from PTX-deficient B. pertussis BPSA87, a strain in which the gene coding for full-length FHA was replaced by that coding for Fha44 [34], failed to induce the secretion of IL-10 by the human MDDC to any measurable level (Fig. 2a).

Please cite this article in press as: Dirix V, et al., Human dendritic cell maturation and cytokine secretion upon stimulation with Bordetella pertussis filamentous haemagglutinin, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.04.003

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Fig. 2. MDDC cytokine secretions induced by different forms of FHA. Cytokine production was measured by ELISA in culture supernatants of MDDC incubated with medium (NS ¼ non-stimulated), 1 mg/ml E. coli LPS or 5 mg/ml of the indicated forms of FHA in the presence of polymyxin B. Bars represent the minimum and maximum values and boxes the medians and 25the75th percentiles of the cytokine concentrations. Significant differences compared to FHA stimulation (*) or to medium only (,) are indicated, */,P < 0.05, **/,,P < 0.01, ***/,,,P < 0.001.

We also tested the different forms of FHA in the presence of polymyxin B for their ability to induce the secretion of other cytokines by human MDDC. Compared to the medium, all forms of FHA were able to significantly induce the secretion of IL-12p40 (Fig. 2b), IL-12p70 (Fig. 2c), IL-23 (Fig. 2d) and IL-6 (Fig. 2e). The levels of IL-12p70 and IL-23 secretion induced by 5 mg/ml of the full-length forms of FHA were similar to those induced by 1 mg/ml of E. coli LPS, whereas the secretion of IL-12p40 and IL-6 induced by the full length FHA forms was slightly lower than those induced by LPS. A slightly, but statistically significantly higher level of IL-12p40 was seen in MDDC culture supernatants after stimulation with FHA purified from ACT-deficient BPSA207 compared to FHA purified from the ACT-producing strain BPRA (Fig. 2b). Compared to the full-length forms of FHA, Fha44 induced significantly lower amounts of all the cytokines tested. However, for all cytokines except IL-10, the levels measured in the MDDC culture supernatants 24 h after stimulation with Fha44 were significantly higher than those found in the supernatants of unstimulated cells. They varied between medians of 2 pg/ml for IL-12p70 to close to 1000 pg/ml for IL-12p40. For IL-23, this difference did not reach statistical significance when a two-tailed test was used, but was statistically different when a one-tailed test was used.

have induced MDDC maturation. We therefore tested the ability of the different forms of FHA to upregulate the maturation markers HLA-DR, CD83, CD40, CD80 and CD86 on the surface of the MDDC. Due to limited amounts of MDDC, the acquisition of a mature phenotype by the MDDC in response to the 4 different molecular forms of FHA was analysed on 15 different MDDC suspensions, each derived from a different blood donor for FHA purified from BPRA in parallel with 6 different cell suspensions, also each derived from a different donor, for FHA purified from BPSA207, 7 for FHARAD and 11 for Fha44. As shown in Fig. 3, immature MDDC shifted to a mature phenotype upon stimulation with FHA, as evidenced by enhanced expression of maturation (HLA-DR, CD83) and costimulatory (CD40, CD80, CD86) molecules. This shift in phenotype was observed regardless of the source of FHA (data not shown for FHA-ACT) and was also observed for FHARAD (Fig. 3). Similar to FHA, the medians of fluorescence intensity (MFI) upon MDDC stimulation with Fha44 were also significantly increased for CD83, HLA-DR, CD40, CD80 and CD86 compared to untreated MDDC (Fig. 4), although Fha44induced expression was always significantly lower than the FHA-induced expression, except for CD40. 4. Discussion

3.3. Phenotypic maturation of MDDC after stimulation with different molecular forms of FHA The cytokine secretion profile of the MDDC stimulated by the different forms of FHA shown above implies that they

FHA is one of the major virulence factors of B. pertussis and has been included in most acellular vaccines. It contains multiple binding domains, including the RGD triplet [28], a CRD [32], an HBD [18] and the MCD [33]. It may thus

Please cite this article in press as: Dirix V, et al., Human dendritic cell maturation and cytokine secretion upon stimulation with Bordetella pertussis filamentous haemagglutinin, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.04.003

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Fig. 3. MDDC maturation induced by FHA, FHA-RAD or LPS. Results of one representative experiment of MDDC cultured 24 h without FHA (grey histograms), with FHA (continuous lines), FHA-RAD (dashed lines) or E. coli LPS (dotted thin lines) are shown. The expression of the indicated surface markers of mature MDDC was analysed by flow cytometry.

interact with different ligands and receptors present on the host respiratory epithelial cells and resident antigen-presenting cells, including alveolar macrophages and dendritic cells (DC). CR3 [28], VLA-5 integrin [31,35] and CD61/CD47 complex [30] have been described as FHA ligands on human epithelial cells and antigen-presenting cells, which may potentially modulate their function and may therefore play a role in the orientation of the immune responses to B. pertussis.

DC are the major antigen-presenting cells and play a key role in the differentiation of naı¨ve T cells into different effector subtypes [36]. As both humoural and Th1-type cellular immune responses contribute to protection against B. pertussis [7,37,38], DC should preferably polarize naı¨ve T cells into the Th1 subset to induce optimal protection against B. pertussis. However, the effect of B. pertussis antigens, such as FHA, on human DC has so far not yet been thoroughly

Fig. 4. Expression of phenotypic maturation markers on MDDC induced by different forms of FHA. MDDC cultured 24 h with medium (NS ¼ non-stimulated), FHA (aee) or Fha44 (fej) in presence of polymyxin B. Values obtained after FHA or Fha44 stimulation are connected with values obtained with medium only for each MDDC obtained from different donors. Horizontal lines represent the medians of median fluorescence intensity (MFI). Significant differences compared to Q6 FHA stimulation (*) or to medium alone (,) are indicated, */,P < 0.05, **/,,P < 0.01, ***/,,,P < 0.001. Please cite this article in press as: Dirix V, et al., Human dendritic cell maturation and cytokine secretion upon stimulation with Bordetella pertussis filamentous haemagglutinin, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.04.003

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investigated. In mice, FHA has previously been reported to express immuno-modulatory activities by inducing the production of IL-10 [14e16], and we have previously shown that in children, the spontaneous secretion of IL-10 by human monocytes correlates with poor Th1-type responses to pertussis vaccines [13]. It was therefore of interest to investigate whether FHA may directly induce immune-modulatory cytokines by human DC and to characterize the in vitro activation of human MDDC by FHA. As PTX is well known for its immune-modulatory functions [39e41], FHA was purified from PTX-deficient strains in order to avoid any possible interference of potentially contaminating PTX with the observed FHA effects on MDDC. We found that FHA is a potent activator of human MDDC and induces the up-regulation of maturation and costimulatory surface molecules, as well as the secretion of IL-10, IL-12p40, IL-12p70, IL-23, and IL-6. The FHAinduced IL-10 secretion has previously been reported for mouse bone marrow-derived DC, but it was not accompanied by IL-12 secretion [15]. Similarly, in mice the upregulation of CD86 and CD40 expression induced by FHA had been observed, but FHA had only a marginal or no effect on HLADR and CD80 expression [15]. However, interference of potentially contaminating LPS could not be ruled out. We have therefore taken special care to avoid any potential interference by LPS using different controls. The LPS content, although variable between different FHA preparations used here, was generally low. In addition, the residual LPS was neutralized by the addition of polymyxin B in the culture medium. The amount of polymyxin B used in this study was validated by preliminary data that established the amount of polymyxin B required to neutralize E. coli LPS for the induction of IL-10. The FHA-induced IL-10 secretion by human DC may thus potentially affect IFN-g responses to B. pertussis antigens, as suggested by our previous study with pertussis-vaccinated infants [13]. However, FHA also induced the secretion of IL-12p70 and IL-23 by MDDC, and these cytokines have been shown to induce Th1 polarization despite high IL-10 levels [40,42], suggesting that a delicate balance between these cytokines is required for an optimal immune response. As ACT can also modulate B. pertussis-induced immune responses [43,44] and is closely associated with FHA on the surface of the bacteria [27], we also tested the effects on human MDDC of FHA purified from a ACT- and PTXdeficient strain. The results obtained with this form of FHA were similar to those obtained with FHA purified from an ACT-producing strain, ruling out a major impact of ACT on the MDDC maturation and cytokine secretion observed here upon stimulation with the FHA preparations. The only exception was a moderate but significant increase in IL-12p40 concentration induced by FHA purified from the ACTdeficient strain compared to FHA purified from the ACTproducing strain, suggesting that a minor contamination by ACT may have diminished the ability of FHA to induce IL12p40. This effect of ACT on the IL-12 secretion is consistent with a previous report showing that ACT can inhibit IL12p40 secretion [43].

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Given the ability of FHA to induce IL-10 secretion by human DC, we wanted to determine whether this property could be physically separated from its ability to induce other cytokines, such as IL-12p70, IL-23 or IL-6. We therefore compared the effect of different molecular forms of FHA on the activation of human MDDC. Among the FHA domains that could potentially affect DC maturation and activity, the RGD sequence was first targeted. This sequence has been reported to bind to the VLA-5 integrin and the CD61/CD47 complex on monocytes, thereby enhancing the CR3 binding activity [30,31]. It has also been reported to directly bind to CR3 on macrophages [28], allowing the entry of the bacteria into these cells, while assuring their intracellular survival [29]. Furthermore, CR3 engagement was reported to inhibit IL-12 secretion [45], and thus affects Th1-type immune responses. As both VLA-5 and CR3 are present on DC, we examined the role of the RGD domain in the FHA-induced maturation and cytokine secretion by MDDC by using FHA-RAD, mutant version of FHA containing a Gly/Ala substitution at the RGD site. This form of FHA induced similar MDDC maturation and cytokine production as native FHA, indicating that the RGD sequence does not play a major role in the FHAinduced activation of human DC in our model. This conclusion is in agreement with previous reports showing no role of the RGD site of FHA in the colonization of the rat respiratory tract by Bordetella bronchiseptica and in the modulation of inflammatory responses in mouse lungs [33]. In contrast to FHA-RAD, the use of Fha44 allowed us to separate the IL-10-inducing properties on MDDC from the IL12-, IL-23-, and IL-6-stimulating activities of FHA. Fha44 lacks the CRD and the MCD, as well as the RGD site of FHA, but contains its HBD domain. Fha44 did not induce any detectable IL-10 secretion by the MDDC, while maintaining the ability to induce IL-12, IL-23 and IL-6 secretions, albeit at lower levels than the full-length forms of FHA. The lack of IL10 secretion by the MDDC in response to stimulation with Fha44 in contrast to full-length FHA could conceivably be due to an overall decreased binding affinity of Fha44, as it lacks several binding domains. However, this is not likely the main reason, as increasing the amount of Fha44 in the assay also does not result in detectable IL-10 secretion (data not shown). Yet it would be expected that lower affinity may be overcome by increasing the ligand concentrations. Phenotypic analyses of Fha44-stimulated MDDC showed that Fha44 induces a maturation profile, which is comparable to that of full-length FHA-stimulated MDDC, as evidenced by the surface markers HLA-DR, CD80, CD83, CD86 and CD40, although the induction of these molecules by Fha44 was significantly lower compared to full-length FHA, except for CD40. The activation of human MDDC by Fha44 may thus be able to drive naı¨ve T cells to Th1-type cells thanks to the IL-12 environment [46] in the absence of IL-10, as well as to Th17-type cells thanks to the secretions of IL-6 and IL-23 known to be important for this T cell polarization in humans [47]. However, this hypothesis requires further investigation. A recent study in humans showed that B. pertussis indeed induces Th17 lymphocytes in addition to Th1 cells [44], and

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studies in mice have demonstrated that these cells play an important role in protective immunity induced by acellular pertussis vaccines [48,49]. Moreover, in some circumstances, such as in Candida albicans infections, Th17 cells can not only produce IL-17 but also IFN-g in an IL-12-independent manner [50]. Thus Fha44 may have the potential to induce Th1-type responses both via IL-12 and via IL-17 in an IL-12independent fashion. A limitation of this study is that we have not been able to confirm the hypothesis that Fha44, in contrast to full-length FHA, can orient the immune response towards a Th1 and Th17 profile. As pertussis vaccination is part of the vaccination program in Belgium, the lymphocytes from adults cultured with Fha44-loaded MDDC would not reflect a primary immune response. This could only be done with blood samples from neonates, before the first pertussis vaccination, which, understandably, would raise important ethical concerns in view of the amount of blood necessary for such experiments. In mice, nasal immunization with Fha44 has been shown to induce strong protection against experimental challenge with virulent B. pertussis [34]. Protection could be transferred to naı¨ve mice by anti-Fha44 antiserum, and at titres similar to anti-full-length FHA titres, provided protection levels similar to those observed after passive transfer of anti-full-length FHA antiserum. At least for mice, Fha44 contains thus the epitope reservoir necessary for protection. These findings, together with our data showing that Fha44 can at least partially induce the maturation of human DC, stimulate the secretion of IL-12, IL-23 and IL-6 by these cells, without the induction of IL-10, suggest that a replacement of full-length FHA by Fha44-like molecules in acellular pertussis vaccines may increase the proportion of children that respond to these vaccines with a strong Th1-type response. We conclude that FHA can modulate human DC maturation and function, which in turn could interfere with the development of appropriate immune responses to acellular vaccine administration and should therefore be taken in consideration. However, these immune-modulatory functions of FHA can be physically separated from its antigenic properties, by using truncated molecules, such as Fha44, which do not express immune-modulatory functions and could therefore be interesting to consider for improving acellular pertussis vaccines. Acknowledgements

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This work was supported by grants from the Belgian Fond de la Recherche Scientifique Me´dicale and the European Marie Curie Host Fellowship. VD was supported by a fellowship from the Fond pour la Formation a` la Recherche dans l’Industrie et dans l’Agriculture and FM was partially supported by a grant from the Fonds National de la Recherche Scientifique. References [1] WHO. Pertussis. WHO; 2013.

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