&get_box_var;

ORIGINAL ARTICLE

CCL17 Production by Dendritic Cells Is Required for NOD1-mediated Exacerbation of Allergic Asthma Saliha Ait Yahia1,2,3,4, Imane Azzaoui1,2,3,4, Laetitia Everaere1,2,3,4, Han Vorng1,2,3,4, Cecile ´ Chenivesse1,2,3,4, 1,2,3,4 1,2,3,4 2,3,4,5 2,3,4,5 Philippe Marquillies , Catherine Duez , Myriam Delacre , Teddy Grandjean , Joanne Balsamelli1,2,3,4, 6,7 1,2,3,4 1,2,3,4 6,7 Martine Fanton d’Andon , Ying Fan , Coline Ple , Catherine Werts , Ivo Gomperts Boneca6,7, 1,2,3,4,8 2,3,4,5 Benoit Wallaert , Mathias Chamaillard , and Anne Tsicopoulos1,2,3,4,8 1

Pulmonary Immunity, Institut National de la Sante´ et de la Recherche Medicale, ´ Lille, France; 2Institut Pasteur de Lille, Center for 3 Infection and Immunity of Lille, Lille, France; Le Centre National de la Recherche Scientifique Unites ´ Mixtes de Recherche, Lille, France; 4Universite´ Lille Nord de France, Lille, France; 5Nods-like receptors in infection and immunity, Institut National de la Sante´ et de la 6 Recherche Medicale, ´ Lille, France; Institut Pasteur, Biologie et Gen ´ etique ´ de la Paroi Bacterienne, Paris, France; 7INSERM, Group Avenir, Paris, France; and 8Clinique des Maladies Respiratoires et Centre Hospitalier Regional ´ et Universitaire de Lille, Lille, France

Abstract Rationale: Pattern recognition receptors are attractive targets for

vaccine adjuvants, and polymorphisms of the innate receptor NOD1 have been associated with allergic asthma. Objectives: To elucidate whether NOD1 agonist may favor allergic asthma in humans through activation of dendritic cells and to evaluate the mechanisms involved using an in vivo model. Methods: NOD1-primed dendritic cells from allergic and

nonallergic donors were characterized in vitro on their phenotype, cytokine secretion, and Th2 polarizing ability. The in vivo relevance was examined in experimental allergic asthma, and the mechanisms were assessed using transfer of NOD1-conditioned dendritic cells from wild-type or CCL17-deficient mice. Measurements and Main Results: NOD1 priming of human dendritic cells promoted a Th2 polarization profile that involved the production of CCL17 and CCL22 in nonallergic subjects but

The mechanisms underlying adjuvant effects are under renewed scrutiny because of the therapeutic potential in vaccine development. Pattern recognition receptors

only CCL17 in allergic patients, without requiring allergen costimulation. Moreover, NOD1-primed dendritic cells from allergic donors exhibited enhanced maturation that led to abnormal CCL22 and IL-10 secretion compared with nonallergic donors. In mice, systemic NOD1 ligation exacerbated allergen-induced experimental asthma by amplifying CCL17-mediated Th2 responses in the lung. NOD1-mediated sensitization of purified murine dendritic cells enhanced production of CCL17 and CCL22, but not of thymic stromal lymphopoietin and IL-33, in vitro. Consistently, adoptive transfer of NOD1-conditioned dendritic cells exacerbated the Th2 pulmonary response in a CCL17-dependent manner in vivo. Conclusions: Data from this study unveil a deleterious role of

NOD1 in allergic asthma through direct induction of CCL17 by dendritic cells, arguing for a need to address vaccine formulation safety issues related to allergy. Keywords: asthma; adjuvant; chemokine; pattern recognition

receptor

are key components for host recognition of danger signals and microorganisms (1) that ultimately shape adaptive immune responses through the activation of

dendritic cells (DCs) and as such are attractive targets for vaccine adjuvants (2). Some pattern recognition receptors, including NOD1, have been shown to favor

( Received in original form October 15, 2013; accepted in final form March 21, 2014 ) This work was supported by Fondation pour la Recherche Medicale ´ (M.C. and S.A.Y.), by ERC starting grant PGNfromSHAPEtoVIR no. 202283 (I.G.B.), and by the Santelys and Societ ´ e´ Franc¸aise d’Allergologie. Author Contributions: S.A.Y. and A.T. designed the research. S.A.Y., I.A., L.E., H.V., J.B., P.M., M.D., T.G., Y.F., C.P., C.W., and M.F.d’A. performed the research. S.A.Y., A.T., M.C., C.D., C.C., I.G.B., and B.W. analyzed the data. S.A.Y., A.T., M.C., and B.W. wrote the paper. Correspondence and requests for reprints should be addressed to Anne Tsicopoulos, Pulmonary Immunity, Center for Infection and Immunity of Lille, Institut Pasteur de Lille, 1 rue du Prof Calmette, BP 245, 59019 Lille, France. E-mail: [email protected] This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org. Am J Respir Crit Care Med Vol 189, Iss 8, pp 899–908, Apr 15, 2014 Copyright © 2014 by the American Thoracic Society Originally Published in Press as DOI: 10.1164/rccm.201310-1827OC on March 24, 2014 Internet address: www.atsjournals.org

Yahia, Azzaoui, Everaere, et al.: NOD1-primed DCs Exacerbate Asthma

899

ORIGINAL ARTICLE

At a Glance Commentary Scientific Knowledge of the Subject: Polymorphisms of NOD1-

encoding gene have been linked to the susceptibility to allergic diseases. However, its functionality in human allergic asthma remains elusive.

DC-derived pro-Th2 chemokine CCL17. Some of the results of these studies have been previously reported in the form of an abstract (15).

Pulmonary Histology

Methods A detailed description of the methods is provided in the online supplement.

What this Study Adds to the Field:

Our data show that NOD1-primed human dendritic cells polarize naive T cells toward a Th2 profile. In experimental asthma, NOD1 acts as a systemic adjuvant and exacerbates allergic Th2 responses through the induction of CCL17 by dendritic cells. Vaccine formulations that engage NOD1 may contribute to increase allergic asthma. not only Th1 (3) or Th17 adaptive responses (4) but also splenic Th2 responses in mice (4), which may lead to susceptibility to developing or exacerbating allergic diseases. NOD1 recognizes peptidoglycan containing mesodiaminopimelic acid (5–7), which is most commonly found in gram-negative bacteria and is primarily expressed in the cytoplasm of nonimmune (8), and immune cells (9), including epithelial and DCs, respectively. In humans, genome-wide association studies have revealed a molecular link between polymorphisms of NOD1, asthma, and high levels of serum IgE (10) as well as with atopic dermatitis (11) and allergic rhinitis (12). NOD1 gene expression changes have been found in nasal mucosa from patients with rhinitis during the pollen season (13). However, the functionality of NOD1 in human allergic asthma remains elusive. It is therefore of utmost importance to determine whether NOD1-mediated adjuvant effects may exacerbate allergic diseases. Given that DCs are central to the initiation of T-cell immunity in allergic asthma (14) and in vaccine immunity (2), the aim of this study was to evaluate if priming of DCs with NOD1 agonist may exacerbate per se a Th2 allergic response in humans and in an experimental model of asthma. Our data demonstrate for the first time a differential responsiveness of DCs from allergic patients to NOD1 agonist together with a proallergenic role of NOD1 through 900

sera by ELISA as previously described (19) with some modifications as described in the online supplement.

Patients

Patients and healthy donors were included as described in the online supplement. Generation of Immature DCs and Maturation

Monocyte-derived DCs (MD-DCs) were generated in vitro from peripheral human blood monocytes as previously described (16) and matured as described in the online supplement. Preparation of Purified T Cells, Proliferation Assay, and DC/T-Cell Cocultures

Allogenic CD41CD45RA1 naive T cells were obtained from peripheral blood mononuclear cells of healthy donors as previously described (17, 18) and stimulated with DCs as indicated in the online supplement.

For lung histology, the left lobe of the lung from each mouse was fixed in Immunohistofix and embedded in resin using the Immunohistowax processing method according to the manufacturer’s indications (Gentaur, Brussels, Belgium). Lung sections of 0.5 mm were stained with a standard hematoxylin-eosin stain and periodic acid-Schiff staining kit (Sigma-Aldrich, St. Louis, MO) to evaluate the peribronchic inflammation and mucopolysaccharide staining, respectively. Measurement of Airway Responsiveness

Airway hyperresponsiveness (AHR) was assessed using Flexivent (Scireq, Montreal, Canada) as previously described (20) and analyzed as described in the online supplement. Lung Protein Extract and Cell Preparations

Lung protein extracts and cells were prepared as described in the online supplement.

Mice

Female C57BL/6J mice (6 wk of age) were purchased from Charles River (Lyon, France) and housed under specific pathogen-free conditions. Nod12/2, ccl172/2, and hNod1 mice are described in the online supplement. Experimental Model of Allergic Asthma

The design of the model is shown in Figure 3A, and further details are available in the online supplement.

Gene Expression Analysis

Lung RNA was extracted as previously described (21) and reverse-transcribed. The resulting cDNA was amplified as previously described (22). Quantification is described in the online supplement. Adoptive Transfer of Antigen-pulsed Bone Marrow–derived DCs

Bronchoalveolar Lavage Fluid Analysis

Bone marrow–derived DCs (BM-DCs) were generated according to a previously published protocol (23). Stimulated BMDCs were transferred as indicated in the online supplement.

Bronchoalveloar lavage (BAL) analysis was performed as described in the online supplement.

Quantification of Chemokine and Cytokine Levels

Measurement of Serum Antibodies

Blood was drawn from the abdominal vein. Serum was collected by centrifugation (300 g for 5 min) and stored at 2208 C until assay. Levels of total IgE, OVA-specific IgE, and IgG1 were measured in collected

Cytokine and chemokine production was measured by ELISA as described in the online supplement. Flow Cytometry Experiments

The cells were stained as described in the online supplement.

American Journal of Respiratory and Critical Care Medicine Volume 189 Number 8 | April 15 2014

ORIGINAL ARTICLE Statistical Analysis

Data were expressed as mean 6 SEM. Statistical methods are described in the online supplement.

Results and Discussion NOD1-primed DCs Promote Th2 Polarization in Allergic and Nonallergic Subjects

To evaluate if NOD1 agonist may potentiate allergic responses in humans, immature human MD-DCs obtained from healthy nonallergic and house dust mite

Dermatophagoides pteronyssinus (Dpt) allergic donors were stimulated with the NOD1 agonist FK565 and/or the major allergen of Dpt Der p 1. The expression of the cell surface costimulatory molecules CD80 and CD86 was significantly enhanced in allergic compared with nonallergic donors upon FK565 stimulation, as was inducible costimulator ligand (ICOSL) expression in allergic donors (Figure 1A). Consistently, NOD1-driven secretion of the regulatory cytokine IL-10 was aborted in DCs from allergic donors when compared with nonallergic individuals (Figure 1B). In contrast, the proinflammatory cytokine IL-6

and chemokine CXCL8 were significantly induced by FK565, as shown in other studies (9, 24) but independently of the allergic status (see Figure E1 in the online supplement). FK565-stimulated DCs from allergic and nonallergic donors produced higher amounts of the pro-Th2 chemokines CCL17 and CCL22 (Figure 1B) than unstimulated DCs, whereas there was no modification of the pro-Th1 chemokine CXCL10 (see Figure E1 in the online supplement). The level of CCL22 was significantly higher in allergic donors than in nonallergic donors. Conversely, the DC immature marker DC-SIGN was

Figure 1. NOD1-primed dendritic cells (DCs) promote Th2 polarization in allergic and nonallergic subjects. (A) Phenotype of human monocyte-derived DCs (MD-DCs) matured with or without NOD1 agonist. Immature MD-DCs from allergic and nonallergic donors were cultured with medium, Der p 1, NOD1 agonist FK565, FK5651Der p 1, and LPS as positive control. DCs were stained with the indicated or matched isotype antibodies and analyzed by flow cytometry. Data are expressed as mean fluorescence intensity (MFI) 6 SEM for 7 to 10 subjects per group. (B) Effect of FK565 on cytokine and chemokine production by mature DCs. DCs were stimulated as described in A, and mediator secretion was quantified by ELISA. Data are expressed as mean 6 SEM for 10 to 14 nonallergic subjects and 8 to 12 allergic subjects. (C) FK565-primed DCs from allergic and nonallergic subjects favor a Th2 profile. DCs activated as described in A were cocultured with naive CD41 T cells for 5 days. The production of polarizing cytokines was analyzed by ELISA in the supernatants. Data are presented as mean 6 SEM for eight nonallergic and seven allergic subjects. *P , 0.05 and **P , 0.01 versus medium; sP , 0.05 and ssP , 0.01 allergic versus nonallergic subjects.

Yahia, Azzaoui, Everaere, et al.: NOD1-primed DCs Exacerbate Asthma

901

ORIGINAL ARTICLE inhibited by FK565 only in nonallergic donors (Figure 1A). In addition, FK565 stimulation did not affect the secretion of TGF-b1 (Figure 1B). The Th2-, Th1-, and Th17-inducing cytokines thymic stromal lymphopoietin (TSLP) and IL-33, IL-12p70, and IL23p19, respectively, were not detected. In line with previous findings, the LPS-positive control increased DC maturation and secretion of all cytokines, regardless of the allergic status, except TGF-b1 and CCL17 (Figure 1B). Stimulation of human DCs from allergic subjects with the LPS-free purified allergen Der p 1 failed to differentially modulate the expression of costimulatory cell surface markers, cytokines, and chemokines (Figures 1A and 1B), indirectly suggesting that Der p 1 may not activate NOD1. These results indicate that NOD1-driven maturation of DCs is further enhanced in allergic patients compared with control subjects. To evaluate whether FK565-stimulated DCs from allergic and nonallergic donors may favor a particular T-cell polarization profile, primed DCs were washed and cocultured with allogenic naive CD41 T cells from nonallergic donors. After 5 days of culture, T-cell polarization was evaluated by measuring the secretion of the Th1 cytokine IFN-g, the Th2 cytokine IL-13, the regulatory cytokines IL-10 and TGF-b, and the Th17 cytokine IL-17A by ELISA. Stimulation of naive T cells with DCs primed with FK565 from allergic and nonallergic donors induced enhanced production of IL-13 but not IFN-g, IL-10, and IL-17A (Figure 1C). To delineate the respective role of CCL17 and CCL22 produced by FK565-primed DCs on the Th2 profile, experiments with neutralizing antibodies against CCL17 and CCL22 were performed. CCL17 and CCL22 were involved in NOD1-driven Th2 polarization in nonallergic subjects, whereas only CCL17 was implicated in NOD1-driven Th2 polarization in allergic patients (Figure E2). To determine the mechanism of action of CCL17 and CCL22 on Th2 polarization, the two chemokines were directly added to DCs. CCL17 slightly increased the percentage of mature CD831 and of CD861 DCs in allergic and nonallergic donors, whereas CCL22 increased it only in nonallergic subjects (Figure 2A). In contrast a strong up-regulation of CD801 and ICOSL1 DCs was observed after CCL17 and CCL22 stimulation in both groups, 902

similar to the level observed for FK565 stimulation (Figure 2A). DCs treated with CCL22 produced more CCL17, whereas DCs treated with CCL17 did not affect CCL22 secretion, suggesting that CCL22 enhances CCL17 production by DCs (Figure 2B). CCL17-treated DCs from both groups induced IL-13 production in T-cell cocultures, whereas CCL22-treated DCs from nonallergic subjects only promoted IL-13 secretion. IFN-g production was not modified (Figure 2C). This effect was independent of T-cell proliferation (Figure 2D) or survival (data not shown) and was not linked to a direct effect of the chemokines on naive T cells (Figure 2E). A polarizing effect of chemokines through DC stimulation has been observed for other chemokines (16, 25, 26). These data show that CCL17 and/or CCL22 produced by NOD1-primed DCs are able to modify DC costimulatory molecule expression and CCL17 secretion and to promote the differentiation of naive CD41 T cells toward a Th2 profile. Systemic NOD1 Elicitation Exacerbates Allergen-induced Cardinal Features of Experimental Asthma

To assess whether the NOD1-driven Th2 response may influence allergic asthma, we set up a model of allergen-induced systemic sensitization without adjuvant followed by intranasal challenges to induce minimal allergic inflammation. Groups of wild-type (WT) C57BL6J mice were sensitized intraperitoneally at 10-day intervals with OVA in combination or not with the NOD1 agonist FK156, which is thought to exhibit higher NF-kB induction activity on murine than human NOD1 (27) (Figure 3A). Control animals received intraperitoneal injections of FK156 or PBS alone. Mice were then challenged intranasally at the indicated time points, with OVA or PBS for the control groups (Figure 3A). The cardinal features of experimental asthma were evaluated in the different groups. WT mice sensitized with OVA alone or with OVA/FK156 and challenged intranasally exhibited an increase in total cell number in BAL when compared with control mice in relation with an increase in lymphocytes and eosinophils in the two groups (Figure 3Ba). Furthermore, total cell, lymphocyte, and eosinophil numbers were significantly higher, whereas macrophage numbers were lower, in OVA/FK156-

sensitized than in OVA-sensitized WT mice. Assessment of the Th2 humoral response showed that the levels of serum total IgE and OVA-specific IgE and IgG1 antibodies were significantly increased in OVA/FK156-sensitized when compared with OVA-sensitized mice (Figure 3Bb). Control experiments showed that the FK565 agonist exhibited proinflammatory activity in mice similar to FK156, although to a lesser extent (Figure E3). In contrast, OVA- and OVA/FK156sensitized Nod12/2 mice exhibited a similar airway eosinophilia and humoral immune response to OVA local challenges compared with their respective controls (Figure 2Bc), thus confirming the NOD1mediated specificity of action of FK156. Previous studies have shown that Th2mediated responses to inhaled OVA require intranasal but not systemic intraperitoneal engagement of TLR4-mediated signaling pathway by low-level LPS in experimental asthma (28, 29). To exclude interference between the adjuvant effect of NOD1 and the low concentration of LPS present in the OVA extracts, Worthington LPS clean OVA (labeled as OVA-clean) was used instead of crude OVA. WT mice sensitized with OVA-clean/FK156 and challenged with OVA-clean showed increased numbers of eosinophils in BAL fluids and increased total IgE production when compared with WT mice sensitized and challenged with OVA-clean alone (Figure 3Bd). Even though the levels of induction were lower in OVA-clean than in crude OVA-sensitized and OVAchallenged WT mice, LPS was not necessary for NOD1-mediated adjuvanticity in experimental allergic asthma but had a synergistic effect on the magnitude of the Th2 allergic response. These results are different from those obtained in a previous study wherein no synergistic effect of LPS was observed on the systemic Th2 response induced by NOD1 (4). This may be related to differences between mucosal and systemic immunity or to the different amount of LPS used during the sensitization process, which was low (0.32 ng) in our model and high (25 mg) in that study. To address this issue, 25 mg of LPS were added during the systemic sensitization with OVA and FK156, and the parameters of the allergic response were assessed in OVA-induced asthma. The addition of a high amount of LPS to FK156 did not exacerbate lung

American Journal of Respiratory and Critical Care Medicine Volume 189 Number 8 | April 15 2014

ORIGINAL ARTICLE

Figure 2. CCL17 and/or CCL22 have a direct effect on dendritic cells (DCs) and favor a Th2 profile. (A) Phenotype of human monocyte-derived DCs (MDDCs) incubated with or without CCL17 or CCL22. Immature MD-DCs from allergic and nonallergic donors were cultured with medium, different concentrations of chemokines, NOD1 agonist FK565, and LPS as positive controls. DCs were stained with the indicated or matched isotype antibodies and analyzed by flow cytometry. Data are expressed as mean percentage of positive cells 6 SEM for 8 to 10 subjects per group. (B) Effect of CCL17 and CCL22 treatment on chemokine production by mature DCs. DCs were stimulated as described in A, and mediator secretion was quantified by ELISA. Data are expressed as mean 6 SEM for 10 nonallergic and 9 allergic subjects. (C) CCL17- and/or CCL22-primed DCs from allergic and nonallergic subjects favor a Th2 profile. DCs activated as described in A were cocultured with naive CD41 T cells for 5 days. The production of polarizing cytokines was analyzed by ELISA in the supernatants. Data are presented as mean 6 SEM for 10 nonallergic and 9 allergic subjects. (D) Chemokine-primed DCs from allergic and nonallergic subjects do not induce T-cell proliferation. CCL17- and CCL22-primed DCs from allergic and nonallergic donors were cocultured with naive T cells labeled with CFSE for 5 days. The percentage of proliferating cells evaluated by decreased CFSE staining was assessed by flow cytometry. Data are presented as mean 6 SEM for 10 nonallergic and 9 allergic subjects. (E) CCL17 and CCL22 do not affect directly the production of IL-13 by T cells. Naive T cells from allergic and nonallergic donors were incubated with different concentrations of chemokines for 5 days. Supernatants were collected and cytokine production assessed by ELISA. Data are presented as mean 6 SEM for 10 nonallergic and 9 allergic subjects. *P , 0.05, **P , 0.01, and ***P , 0.001 versus medium; ssP , 0.01 allergic versus nonallergic subjects.

OVA-induced Th2 responses, suggesting that the difference between the two studies is linked to LPS concentration (Figure E4). Histological analysis showed increased inflammatory infiltrates and mucus staining in crude OVA/FK156-sensitized as compared with OVA-sensitized WT mice (Figure 3C). Consistently, OVA challenge of OVA-sensitized mice induced a significant increase in AHR compared with control mice (Figure 3D). OVA/ FK156-sensitized mice demonstrated a higher increase in AHR than OVA-, FK156-, and PBS-sensitized mice. To evaluate the polarization profile, Th2-type

and pro-Th2 chemokine concentrations were measured in lung protein extracts by ELISA. In line with our previous results, IL-13 (Figure 3E) and IL-4 (Figure E5A) were significantly increased after sensitization with OVA/FK156 when compared with sensitization with OVA alone in WT mice. Consistently, pro-Th2 chemokine CCL17 and CCL22 protein and mRNA levels were higher in WT mice sensitized with OVA/ FK156 than in mice sensitized with OVA alone (Figures 3E and E5B). Among the pro-eosinophilic mediators, IL-5 mRNA expression was increased in OVA- and OVA/FK156-sensitized mice as compared

Yahia, Azzaoui, Everaere, et al.: NOD1-primed DCs Exacerbate Asthma

with PBS-treated control mice, whereas CCL5, a chemokine induced in murine macrophages by NOD1 stimulation (30), was not significantly modified (Figure E5B). There was no variation of TSLP, whereas IL-33 increased similarly in OVA- and OVA/FK156-sensitized mice (Figure 3E). In contrast, no significant changes were observed for the Th1 cytokine IFN-g (Figure 3E). In addition, the levels of the pro-Th1 chemokine CXCL10 and of the Th17 cytokine IL-17A decreased in OVAsensitized mice (Figure E5A). We next investigated the cellular sources responsible for the increased 903

ORIGINAL ARTICLE

Figure 3. Systemic NOD1 elicitation exacerbates allergen-induced cardinal features of experimental asthma. (A) Experimental design. Mice were sensitized at Days 0 and 10 by intraperitoneal injection of OVA with or without NOD1 agonist FK156. Mice were challenged intranasally with 100 mg of OVA as indicated and killed on Day 34. Control mice were immunized with PBS alone or with PBS1FK156 and challenged with PBS only. (B) Effects of NOD1 priming on BAL cell counts and humoral responses. (a) BAL cell counts in wild-type (WT) mice. (b) Levels of total IgE antibody and OVA-specific IgE (titer) and IgG1 antibodies (titer 3106) in WT mice. (c) BAL cell counts and total IgE antibodies in nod12/2 mice. (d) BAL cell counts and total IgE antibodies in WT mice sensitized and challenged with OVA-clean. Data are expressed as mean 6 SEM (n = 12–16 per group for a and b, n = 8 for c, and n = 5–6 for d). Results are from two or three independent experiments. (C) Representative microphotographs of periodic acid Schiff (PAS)-stained and hematoxylin and eosin (H&E)-stained lung sections (original magnification: 3x312.5) in WT mice. Scale bar: 100 mm. (D) Airway hyperresponsiveness of WT mice. Results are expressed as percentage of lung resistance increase above PBS and presented as mean 6 SEM (n = 5–10 from two independent experiments). (E) Cytokine and chemokine production in lung protein extracts from WT mice determined by ELISA. Data are expressed as mean 6 SEM (n = 8 –10) in each group. Results are from two independent experiments. For all graphs: *P , 0.05, **P , 0.01, and ***P , 0.001 versus control mice treated with either PBS or FK156; # P , 0.05 and ##P , 0.01 for OVA1FK156/OVA versus OVA/OVA-treated mice. TSLP = thymic stromal lymphopoietin.

production of CCL17 and CCL22 in the lung of mice sensitized with OVA/FK156 by intracellular flow cytometry staining. CCL17 and CCL22 are produced by many different lung cell types, including cytokeratin1 epithelial cells, MHCII1/CD11c1 DCs, F4/801 macrophages, CD191 B cells, and 904

CD41 T cells (31–34). All lung cell types except B cells were found to produce the two chemokines. The number of CCL17-secreting DCs, CD41 T cells, and macrophages was significantly increased after OVA/FK156 sensitization compared with the OVA group, whereas the number

of CCL17-producing epithelial cells was not modified (Figure E6A). For CCL22, only the number of CCL22-producing macrophages was increased in OVA/ FK156-sensitized mice (Figure E6A). When evaluating the percentage of positive cells among the different cell populations, only

American Journal of Respiratory and Critical Care Medicine Volume 189 Number 8 | April 15 2014

ORIGINAL ARTICLE CD41 T cells exhibited a significant increase in the percentage of CCL17producing cells in OVA/FK156-sensitized compared with OVA-sensitized mice (34.03 6 2.99% vs. 23.55 6 2.62%; P , 0.05; n = 4 experiments), whereas CCL22 was produced by a limited percentage of CD41 T cells. Representative flow cytometry staining of CCL17 and CCL22 in CD41 T cells is shown in Figure E6B. These data suggest that, in addition to increased lung cell recruitment, a specific priming of T cells has occurred by cells systemically activated by NOD1, leading to increased CCL17 production. Altogether, these data demonstrate that NOD1, used as a systemic adjuvant, promotes lung Th2 asthmatic reaction in mice. CCL17 Production by NOD1conditioned DCs Is Required to Exacerbate Experimental Asthma

Our human findings pointed toward a role for NOD1-primed DCs in the exacerbation of Th2 polarization. To assess the role of DCs in NOD1-driven exacerbation of

allergic experimental asthma, adoptive transfer experiments were performed. BMDCs were pulsed with OVA in the presence or absence of FK156. After washing, DCs were transferred intraperitoneally to naive WT mice that were then challenged with OVA. As reported by others for intratracheal transfer (29, 35), intraperitoneal transfer of OVA-pulsed BM-DCs reconstituted Th2 cell responses to inhaled antigen. Transfer of DCs pulsed with OVA/FK156 induced a significant increase in airway total cell number and in the numbers of eosinophils and lymphocytes in BAL compared with the transfer of DCs pulsed with OVA or PBS alone (Figure 4Aa). Increased production of total IgE and of OVA-specific IgE and IgG1 antibodies was observed in serum from mice transferred with DCs pulsed with OVA/FK156 when compared with those transferred with DCs pulsed with OVA or with PBS alone (Figure 4Ab). Lung Th2 polarization was also increased in mice transferred with OVA/FK156-pulsed DCs compared with the other groups, as shown

by the elevated expression of IL-13 and IL-5 mRNA and of CCL17 at the protein level (Figure 4Ac). No effects were observed with the transfer of FK-156–pulsed DCs on either parameter (data not shown). In agreement with our previous results, transfer of OVA/LPS 25 mg/FK156–pulsed DCs did not exacerbate OVA-induced responses (Figure E7). These results suggest that the adjuvant effect of NOD1 agonist on Th2 lung polarization in OVA-induced asthma is dependent upon the direct activation of DCs by NOD1. There was no induction of TSLP and IL-33 in DC supernatants, whereas CCL17 and CCL22 secretion was up-regulated in OVA/FK156pulsed DCs compared with DCs pulsed with OVA or PBS alone but not in FK156pulsed DCs (Figure 5A). This last result is in contrast with the data obtained in humans, where the effect of NOD1 agonist on the production of pro-Th2 chemokines did not require the presence of the allergen. This difference was not related to the use of two different NOD1 ligands because FK565 behaved similarly to FK156

Figure 4. CCL17 production by NOD1-conditioned dendritic cells (DCs) is required to exacerbate experimental asthma. Bone marrow–derived DCs (BM-DCs) from wild-type (WT) mice (A) or ccl172/2 mice (B) were pulsed in vitro with PBS alone or with OVA with or without FK156, washed, and transferred intraperitoneally on Day 1 into naive WT mice. Mice were challenged with OVA from Day 7 to 9 and from Day14 to 16. Mice were killed 96 hours later, and BAL and serum were collected. (a) BAL cell counts. (b) Levels of total IgE, OVA-specific IgE (titer), and IgG1 antibodies (titer 3106). (c) Protein and mRNA expression of chemokine and cytokines. Data are expressed as mean 6 SEM (n = 6 per group for WT DCs; n = 4–5 for ccl172/2 DCs). *P , 0.05 and **P , 0.01 versus control mice treated with PBS. #P , 0.05 and ##P , 0.01 for OVA1FK156/OVA versus OVA/OVA-treated mice. ND = not detectable; ns = not significant.

Yahia, Azzaoui, Everaere, et al.: NOD1-primed DCs Exacerbate Asthma

905

ORIGINAL ARTICLE

Figure 5. In vitro production of CCL17 and CCL22 by pulsed bone marrow–derived dendritic cells (BM-DCs). Cell supernatants from BM-DCs derived from wild-type mice (A), hNod1 mice (B), and Nod12/2 mice (C) and stimulated as indicated were recovered and analyzed by ELISA. Results are expressed as mean 6 SEM (n = 4–10 in each group). *P , 0.05 and **P , 0.001 versus control BM-DCs treated with PBS; #P , 0.05 for OVA1FK versus OVA BM-DCs.

(Figure 5A). Dose-response experiments showed that this difference was not related to FK156 concentration (Figure E8). Because NOD1 sensing has been shown to differ between mice and humans (36), we generated a transgenic mouse model expressing only the human NOD1encoding gene (hNod1) to evaluate potential differences in DC species–specific responsiveness to FK156 and FK565. In the absence of OVA stimulation, as observed in human DCs, FK156- and FK565-pulsed DCs from hNod1 mice produced significantly increased levels of CCL17 and CCL22 (Figure 5B), whereas none of these chemokines was secreted by DCs from Nod12/2 mice (Figure 5C). Collectively, our data suggest that species-specific mechanisms may partly influence the responsiveness to NOD1 agonists. To link the production of pro-Th2 chemokines by NOD1-stimulated DCs with the amplification of the lung Th2 profile in OVA-induced asthma, OVA and/or FK156pulsed DCs from ccl172/2 mice were transferred to naive WT mice. Transfer of ccl172/2 DCs pulsed with OVA/FK156 induced a significant increase in airway

total cell number and in eosinophils and lymphocytes in the BAL, which was not different from mice transferred with ccl172/2 DCs pulsed with OVA alone (Figure 4Ba). Total serum IgE and OVAspecific IgG1 antibodies also did not differ between the two groups of mice, whereas OVA-specific IgE antibodies were not detected (Figure 4Bb). Similarly, lung Th2 cytokine and CCL17 levels were not different between mice transferred with OVA/FK156- and OVA-pulsed ccl172/2 DCs (Figure 4Bc). Finally, no difference was also observed at the histopathology level (data not shown). Although an increase in BAL total cell and lymphocyte numbers was observed in OVA-pulsed ccl172/2 DCs compared with OVA-pulsed WT DCs, all the specific Th2 endpoints were similar. This absence of a difference in Th2 endpoints suggests that, although CCL22 was also induced in OVA/FK156stimulated DCs, it did not play a major role in the exacerbation of allergic inflammation. These data suggest that CCL17 produced by DCs is required for NOD1-mediated amplification of OVAinduced lung Th2 polarization in mice. The importance of CCL17 production by DCs

References 1. Opitz B, van Laak V, Eitel J, Suttorp N. Innate immune recognition in infectious and noninfectious diseases of the lung. Am J Respir Crit Care Med 2010;181:1294–1309.

906

in Th2 polarization has been stressed in previous studies in allergic asthma (37) and in a humanized SCID mouse model of asthma (38). CCL17 production by DCs during the sensitization process may lead to increased duration of interaction between CCR4-expressing CD41 T cells and NOD1stimulated DCs and thus reinforce cytokine production by the antigen-primed T cells. Such a mechanism has been described for DCs infected by Listeria monocytogenes (39). Moreover, it has been shown that DCs producing CCL17 are better stimulators of CD41 T-cell proliferation than ccl172/2 DCs (40). In the aforementioned work, splenic DCs expressed almost no CCL17, in contrast to other lymphoid and nonlymphoid tissue DCs, which may explain the difference of mechanisms observed between our study and a previously published work (41). Indeed, in that study, the adjuvant effect of NOD1 for splenic Th2 responses was ascribed to TSLP produced by stromal cells, whereas our findings demonstrate a key role of DCderived CCL17 for NOD1-driven lung Th2 responses. These results further support the idea that the mechanism of Th2 amplification by NOD1 agonist differs according to the target tissue. In this context it is of interest that, although NOD1 and NOD2 agonists given systemically are able to favor a splenic Th2mediated adaptive response (4), only NOD2 was shown to favor Th2 allergic inflammation when administered locally in the lung (42). These results identify a novel unrecognized target downstream of NOD1 in human DCs, which is likely to play a key role in orchestrating the global Th2 response in particular in the development and/or exacerbation of allergic asthma, and stress the importance of addressing safety issues linked to allergic diseases for vaccine formulations that engage NOD1, which may contribute to an increase in the global burden of allergic asthma. n Author disclosures are available with the text of this article at www.atsjournals.org.

2. Levitz SM, Golenbock DT. Beyond empiricism: informing vaccine development through innate immunity research. Cell 2012;148: 1284–1292. 3. Rastogi R, Du W, Ju D, Pirockinaite G, Liu Y, Nunez G, Samavati L. Dysregulation of p38 and MKP-1 in response to NOD1/TLR4

American Journal of Respiratory and Critical Care Medicine Volume 189 Number 8 | April 15 2014

ORIGINAL ARTICLE stimulation in sarcoid bronchoalveolar cells. Am J Respir Crit Care Med 2011;183:500–510. 4. Fritz JH, Le Bourhis L, Sellge G, Magalhaes JG, Fsihi H, Kufer TA, Collins C, Viala J, Ferrero RL, Girardin SE, et al. Nod1-mediated innate immune recognition of peptidoglycan contributes to the onset of adaptive immunity. Immunity 2007;26:445–459. 5. Chamaillard M, Girardin SE, Viala J, Philpott DJ. Nods, Nalps and Naip: intracellular regulators of bacterial-induced inflammation. Cell Microbiol 2003;5:581–592. 6. Girardin SE, Boneca IG, Carneiro LA, Antignac A, Jehanno ´ M, Viala J, Tedin K, Taha MK, Labigne A, Zahringer ¨ U, et al. Nod1 detects a unique muropeptide from gram-negative bacterial peptidoglycan. Science 2003;300:1584–1587. 7. Cartwright N, Murch O, McMaster SK, Paul-Clark MJ, van Heel DA, Ryffel B, Quesniaux VF, Evans TW, Thiemermann C, Mitchell JA. Selective NOD1 agonists cause shock and organ injury/dysfunction in vivo. Am J Respir Crit Care Med 2007;175:595–603. 8. Uehara A, Fujimoto Y, Fukase K, Takada H. Various human epithelial cells express functional Toll-like receptors, NOD1 and NOD2 to produce anti-microbial peptides, but not proinflammatory cytokines. Mol Immunol 2007;44:3100–3111. 9. Fritz JH, Girardin SE, Fitting C, Werts C, Mengin-Lecreulx D, Caroff M, Cavaillon JM, Philpott DJ, Adib-Conquy M. Synergistic stimulation of human monocytes and dendritic cells by Toll-like receptor 4 and NOD1- and NOD2-activating agonists. Eur J Immunol 2005;35: 2459–2470. 10. Hysi P, Kabesch M, Moffatt MF, Schedel M, Carr D, Zhang Y, Boardman B, von Mutius E, Weiland SK, Leupold W, et al. NOD1 variation, immunoglobulin E and asthma. Hum Mol Genet 2005;14: 935–941. 11. Weidinger S, Klopp N, Rummler L, Wagenpfeil S, Novak N, Baurecht HJ, Groer W, Darsow U, Heinrich J, Gauger A, et al. Association of NOD1 polymorphisms with atopic eczema and related phenotypes. J Allergy Clin Immunol 2005;116:177–184. 12. Eder W, Klimecki W, Yu L, von Mutius E, Riedler J, Braun-Fahrlander ¨ C, Nowak D, Holst O, Martinez FD; ALEX-Team. Association between exposure to farming, allergies and genetic variation in CARD4/NOD1. Allergy 2006;61:1117–1124. 13. Bogefors J, Rydberg C, Uddman R, Fransson M, Mansson ˚ A, Benson M, Adner M, Cardell LO. Nod1, Nod2 and Nalp3 receptors, new potential targets in treatment of allergic rhinitis? Allergy 2010;65: 1222–1226. 14. Lambrecht BN, Hammad H. Biology of lung dendritic cells at the origin of asthma. Immunity 2009;31:412–424. 15. Ait Yahia S, Azzaoui I, Vorng H, Marquillies P, Duez C, Delacre M, Grandjean T, Glineur C, Everaere L, Fan Y, et al. CCL17 production by dendritic cells is required for NOD1-mediated exacerbation of allergic asthma [abstract]. Presented at the Keystone Symposium: Type 2 immunity and pathogenic processes in asthma and COPD, Santa Fe, NM, January 10–15, 2013. 16. Azzaoui I, Yahia SA, Chang Y, Vorng H, Morales O, Fan Y, Delhem N, Ple C, Tonnel AB, Wallaert B, et al. CCL18 differentiates dendritic cells in tolerogenic cells able to prime regulatory T cells in healthy subjects. Blood 2011;118:3549–3558. 17. Chang Y, de Nadai P, Azzaoui I, Morales O, Delhem N, Vorng H, Tomavo S, Ait Yahia S, Zhang G, Wallaert B, et al. The chemokine CCL18 generates adaptive regulatory T cells from memory CD41 T cells of healthy but not allergic subjects. FASEB J 2010;24: 5063–5072. 18. Chenivesse C, Chang Y, Azzaoui I, Ait Yahia S, Morales O, Ple´ C, Foussat A, Tonnel AB, Delhem N, Yssel H, et al. Pulmonary CCL18 recruits human regulatory T cells. J Immunol 2012;189: 128–137. 19. Biet F, Duez C, Kremer L, Marquillies P, Amniai L, Tonnel AB, Locht C, Pestel J. Recombinant Mycobacterium bovis BCG producing IL-18 reduces IL-5 production and bronchoalveolar eosinophilia induced by an allergic reaction. Allergy 2005;60:1065–1072. 20. Ple C, Barrier M, Amniai L, Marquillies P, Bertout J, Tsicopoulos A, Walzer T, Lassalle P, Duez C. Natural killer cells accumulate in lungdraining lymph nodes and regulate airway eosinophilia in a murine model of asthma. Scand J Immunol 2010;72:118–127.

21. Sen ´ echal ´ S, de Nadai P, Ralainirina N, Scherpereel A, Vorng H, Lassalle P, Tonnel AB, Tsicopoulos A, Wallaert B. Effect of diesel on chemokines and chemokine receptors involved in helper T cell type 1/type 2 recruitment in patients with asthma. Am J Respir Crit Care Med 2003;168:215–221. 22. Normand S, Delanoye-Crespin A, Bressenot A, Huot L, Grandjean T, Peyrin-Biroulet L, Lemoine Y, Hot D, Chamaillard M. Nod-like receptor pyrin domain-containing protein 6 (NLRP6) controls epithelial self-renewal and colorectal carcinogenesis upon injury. Proc Natl Acad Sci USA 2011;108:9601–9606. 23. Ryman KD, White LJ, Johnston RE, Klimstra WB. Effects of PKR/RNase L-dependent and alternative antiviral pathways on alphavirus replication and pathogenesis. Viral Immunol 2002;15:53–76. 24. Tada H, Aiba S, Shibata K, Ohteki T, Takada H. Synergistic effect of Nod1 and Nod2 agonists with toll-like receptor agonists on human dendritic cells to generate interleukin-12 and T helper type 1 cells. Infect Immun 2005;73:7967–7976. 25. Cappello P, Fraone T, Barberis L, Costa C, Hirsch E, Elia AR, Caorsi C, Musso T, Novelli F, Giovarelli M. CC-chemokine ligand 16 induces a novel maturation program in human immature monocytederived dendritic cells. J Immunol 2006;177:6143–6151. 26. Marsland BJ, Battig ¨ P, Bauer M, Ruedl C, Lassing ¨ U, Beerli RR, Dietmeier K, Ivanova L, Pfister T, Vogt L, et al. CCL19 and CCL21 induce a potent proinflammatory differentiation program in licensed dendritic cells. Immunity 2005;22:493–505. 27. Magalhaes JG, Philpott DJ, Nahori MA, Jehanno ´ M, Fritz J, Le Bourhis L, Viala J, Hugot JP, Giovannini M, Bertin J, et al. Murine Nod1 but not its human orthologue mediates innate immune detection of tracheal cytotoxin. EMBO Rep 2005;6:1201–1207. 28. Eisenbarth SC, Piggott DA, Huleatt JW, Visintin I, Herrick CA, Bottomly K. Lipopolysaccharide-enhanced, toll-like receptor 4-dependent T helper cell type 2 responses to inhaled antigen. J Exp Med 2002;196: 1645–1651. 29. Piggott DA, Eisenbarth SC, Xu L, Constant SL, Huleatt JW, Herrick CA, Bottomly K. MyD88-dependent induction of allergic Th2 responses to intranasal antigen. J Clin Invest 2005;115:459–467. 30. Werts C, le Bourhis L, Liu J, Magalhaes JG, Carneiro LA, Fritz JH, Stockinger S, Balloy V, Chignard M, Decker T, et al. Nod1 and Nod2 induce CCL5/RANTES through the NF-kappaB pathway. Eur J Immunol 2007;37:2499–2508. 31. Penna G, Vulcano M, Roncari A, Facchetti F, Sozzani S, Adorini L. Cutting edge: differential chemokine production by myeloid and plasmacytoid dendritic cells. J Immunol 2002;169:6673–6676. 32. Sekiya T, Miyamasu M, Imanishi M, Yamada H, Nakajima T, Yamaguchi M, Fujisawa T, Pawankar R, Sano Y, Ohta K, et al. Inducible expression of a Th2-type CC chemokine thymus- and activationregulated chemokine by human bronchial epithelial cells. J Immunol 2000;165:2205–2213. 33. Nakayama T, Hieshima K, Nagakubo D, Sato E, Nakayama M, Kawa K, Yoshie O. Selective induction of Th2-attracting chemokines CCL17 and CCL22 in human B cells by latent membrane protein 1 of Epstein-Barr virus. J Virol 2004;78:1665–1674. 34. Hirata H, Arima M, Cheng G, Honda K, Fukushima F, Yoshida N, Eda F, Fukuda T. Production of TARC and MDC by naive T cells in asthmatic patients. J Clin Immunol 2003;23:34–45. 35. Lambrecht BN, Pauwels RA, Fazekas De St Groth B. Induction of rapid T cell activation, division, and recirculation by intratracheal injection of dendritic cells in a TCR transgenic model. J Immunol 2000;164: 2937–2946. 36. Girardin SE, Jehanno ´ M, Mengin-Lecreulx D, Sansonetti PJ, Alzari PM, Philpott DJ. Identification of the critical residues involved in peptidoglycan detection by Nod1. J Biol Chem 2005;280: 38648–38656. 37. Medoff BD, Seung E, Hong S, Thomas SY, Sandall BP, Duffield JS, Kuperman DA, Erle DJ, Luster AD. CD11b1 myeloid cells are the key mediators of Th2 cell homing into the airway in allergic inflammation. J Immunol 2009;182:623–635. 38. Perros F, Hoogsteden HC, Coyle AJ, Lambrecht BN, Hammad H. Blockade of CCR4 in a humanized model of asthma reveals a critical role for DC-derived CCL17 and CCL22 in attracting Th2 cells and inducing airway inflammation. Allergy 2009;64:995–1002.

Yahia, Azzaoui, Everaere, et al.: NOD1-primed DCs Exacerbate Asthma

907

ORIGINAL ARTICLE 39. Henry CJ, Ornelles DA, Mitchell LM, Brzoza-Lewis KL, Hiltbold EM. IL12 produced by dendritic cells augments CD81 T cell activation through the production of the chemokines CCL1 and CCL17. J Immunol 2008;181:8576–8584. 40. Alferink J, Lieberam I, Reindl W, Behrens A, Weiss S, Huser ¨ N, Gerauer K, Ross R, Reske-Kunz AB, Ahmad-Nejad P, et al. Compartmentalized production of CCL17 in vivo: strong inducibility in peripheral dendritic cells contrasts selective absence from the spleen. J Exp Med 2003; 197:585–599.

908

41. Magalhaes JG, Rubino SJ, Travassos LH, Le Bourhis L, Duan W, Sellge G, Geddes K, Reardon C, Lechmann M, Carneiro LA, et al. Nucleotide oligomerization domain-containing proteins instruct T cell helper type 2 immunity through stromal activation. Proc Natl Acad Sci USA 2011;108:14896–14901. 42. Duan W, Mehta AK, Magalhaes JG, Ziegler SF, Dong C, Philpott DJ, Croft M. Innate signals from Nod2 block respiratory tolerance and program T(H)2-driven allergic inflammation. J Allergy Clin Immunol 2010;126:1284–1293, e10.

American Journal of Respiratory and Critical Care Medicine Volume 189 Number 8 | April 15 2014

CCL17 production by dendritic cells is required for NOD1-mediated exacerbation of allergic asthma.

Pattern recognition receptors are attractive targets for vaccine adjuvants, and polymorphisms of the innate receptor NOD1 have been associated with al...
2MB Sizes 2 Downloads 3 Views