Increased Th2 Cytokine Secretion, Eosinophilic Airway Inflammation, and Airway Hyperresponsiveness in Neurturin-Deficient Mice This information is current as of September 28, 2021. Tatiana Michel, Maud Thérésine, Aurélie Poli, Olivia Domingues, Wim Ammerlaan, Nicolaas H. C. Brons, François Hentges and Jacques Zimmer J Immunol 2011; 186:6497-6504; Prepublished online 20 April 2011; Downloaded from doi: 10.4049/jimmunol.1001673 http://www.jimmunol.org/content/186/11/6497 http://www.jimmunol.org/ References This article cites 34 articles, 8 of which you can access for free at: http://www.jimmunol.org/content/186/11/6497.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Increased Th2 Cytokine Secretion, Eosinophilic Airway Inflammation, and Airway Hyperresponsiveness in Neurturin-Deficient Mice

Tatiana Michel,* Maud The´re´sine,* Aure´lie Poli,* Olivia Domingues,* Wim Ammerlaan,† Nicolaas H. C. Brons,† Franc¸ois Hentges,* and Jacques Zimmer*

Neurotrophins such as nerve and brain-derived neurotrophic factor have been described to be involved in the path- ogenesis of asthma. Neurturin (NTN), another from the glial cell line-derived neurotrophic factor family, was shown to be produced by human immune cells: monocytes, B cells, and T cells. Furthermore, it was previously described that the secretion of inflammatory cytokines was dramatically stimulated in NTN knockout (NTN2/2) mice. NTN is structurally similar to TGF-b,

a protective cytokine in airway inflammation. This study investigates the implication of NTN in a model of allergic airway Downloaded from inflammation using NTN2/2 mice. The bronchial inflammatory response of OVA-sensitized NTN2/2 mice was compared with wild-type mice. Airway inflammation, Th2 cytokines, and airway hyperresponsiveness (AHR) were examined. NTN2/2 mice showed an increase of OVA-specific serum IgE and a pronounced worsening of inflammatory features. Eosinophil number and IL-4 and IL-5 concentration in the bronchoalveolar lavage fluid and lung tissue were increased. In parallel, Th2 cytokine secretion of lung draining lymph node cells was also augmented when stimulated by OVA in vitro. Furthermore, AHR was markedly 2/2 enhanced in NTN mice after sensitization and challenge when compared with wild-type mice. Administration of NTN before http://www.jimmunol.org/ challenge with OVA partially rescues the phenotype of NTN2/2 mice. These findings provide evidence for a dampening role of NTN on allergic inflammation and AHR in a murine model of asthma. The Journal of Immunology, 2011, 186: 6497–6504. llergic asthma is characterized by an inflammatory air- nergic noncholinergic nervous system, through the release of way obstruction triggered by inhalation of specific neuropeptides such as substance P, contributes to airway smooth A allergens. A hallmark of allergic asthma is an inap- muscle contraction and to airway hyperresponsiveness (AHR) and propriate regulation of the Th1/Th2 balance in favor of a Th2 modulates neurotrophin synthesis (2). are mole- response. Asthmatic airways are characterized by infiltrates of eo- cules originally described for their effects on neuronal cells. They

sinophils and T lymphocytes secreting Th2 cytokines: IL-4, IL-5, control differentiation, development, and survival of neurons (3, by guest on September 28, 2021 and IL-13 (1). The production of IL-5 results in maturation and 4). Factors with a neurotrophic activity can be divided into three enhanced recruitment of eosinophils into airways mucosa. Asthma structurally different families: 1) the (NGF) may result in acute life-threatening reactions but more commonly family including NGF, brain-derived neurotrophic factor (BDNF), results in a chronic disabling disease. Many efforts have been neurotrophin-3, and neurotrophin-4; 2) the ciliary neurotrophic spent in the last 10 years to characterize the regulatory events factor/IL-6 family; and 3) the glial cell line-derived neurotrophic involved in the balance of Th1/Th2 cytokines, to identify signif- factor (GDNF) family, including GDNF, neurturin (NTN), arte- icant therapeutic targets. An effective therapy for asthma would be min, and . Neurotrophins such as NGF and BDNF are to inhibit the development of such allergen-specific Th2 cells, but expressed in a variety of nonneural tissues, including the pulmo- the factors responsible for the initial induction of the Th2-like nary system (5). Recent studies suggest that NGF and BDNF response in vivo remain incompletely understood. participate in the pathogenesis of many features and symptoms of Neural regulation of the airways consists of cholinergic, ad- asthma (2, 6–8). renergic, and nonadrenergic noncholinergic nerves. The nonadre- Intensive research has focused on NGF, which is the prominent member of the neurotrophin family; however, several recent reports suggest that GDNF family members may also modulate airway *Laboratoire d9Immunoge´ne´tique et d9Allergologie, Centre de Recherche Public de inflammation. It was described that GDNF is expressed in mouse la Sante´, L-1526 Luxembourg, Luxembourg; and †Platforme de Cytometrie en Flux, lung, in particular in airway smooth muscle cells. After allergen Centre de Recherche Public de la Sante´, L-1526 Luxembourg, Luxembourg challenge in OVA-sensitized mice, the number of GDNF+ cells was Received for publication May 19, 2010. Accepted for publication March 14, 2011. shown to increase within the inflammatory infiltrate (9). Another This work was supported by a grant from the Fonds National de la Recherche, member of this family, NTN, exerts its effects through a multi- Luxembourg. component receptor system consisting of the GDNF family re- Address correspondence and reprint requests to Dr. Tatiana Michel and Dr. Jacques Zimmer, Laboratoire d9Immunoge´ne´tique et d9Allergologie, Centre de Recherche ceptor a 2 (GFRa-2) and the proto-oncogene RET (10, 11). It was Public CRP-Sante´, 84 Val Fleuri, L-1526 Luxembourg, Luxembourg. E-mail previously shown that NTN is produced by human immune cells: addresses: [email protected] and [email protected] monocytes, B cells, and T cells (12). NTN is also structurally Abbreviations used in this article: AHR, airway hyperresponsiveness; BALF, bron- b b choalveolar lavage fluid; BDNF, brain-derived neurotrophic factor; CBA, Cytometric similar to TGF- , and it was demonstrated that TGF- 1 is a Bead Array; Cdyn, dynamic compliance; GDNF, glial cell line-derived neurotrophic protective cytokine in airway inflammation (13). Furthermore, it factor; GFRa-2, GDNF family receptor a 2; LN, lymph node; NGF, nerve growth was described that the production of inflammatory cytokines was factor; NTN, neurturin; PAS, periodic acid-Schiff; WT, wild-type. dramatically stimulated in the corneal epithelia of the NTN 2/2 Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 knockout (NTN ) mice. These mice also have an immune www.jimmunol.org/cgi/doi/10.4049/jimmunol.1001673 6498 NEURTURIN INVOLVED IN ALLERGIC ASTHMA

methacholine. Before testing, mice were anesthetized by s.c. injection of 80 mg/kg ketamine, 10 mg/kg xylazine, and 2.5 mg/kg midazolam. Then mice were tracheotomized, mechanically ventilated, and lung function was an- alyzed by Flexivent equipment (Emka Technologies, Paris, France). Bronchoalveolar lavage fluid, lung, and lymph node cell isolation and analysis

FIGURE 1. Expression of GFRa-2 mRNA by RT-PCR. Tissues from Immediately after assessment of AHR, lungs were washed via a tracheal 3 WT brain, lung, and lung draining LN were prepared. The experiments tube with PBS and 5% BSA (700 ml) followed by PBS (2 500 ml). Lung cells were isolated using collagenase digestion, followed by staining for were performed by using primers for GFRa-2 and GAPDH as a house- FACS analysis. Lung draining lymph nodes (LNs) were excised from mice, keeping gene. There were three mice in each group. B, brain; L, lung; W, and cells were isolated, adjusted to 5 3 106 cells/ml, and cultured in 48- water. well plates in DMEM supplemented with 10% FBS, 100 U/ml penicillin/ streptomycin, and IL-2 (1000 U/ml) for 1 or 3 d with or without OVA (200 activation of the conjunctival epithelium and an abnormal gut mg/ml). Bronchoalveolar lavage fluid (BALF), lung, and lung draining LN 2 motility caused by a reduction in substance P secretion by gut supernatants were frozen at 80˚C for later cytokine analysis. All cell culture reagents were purchased from Invitrogen (Paisley, U.K.). muscle cells (14, 15). In this study, we assessed the influence of 2/2 NTN on lung function. NTN mice were investigated for airway Measurement of OVA-specific Abs inflammation and for development of AHR after sensitization and Before performing BALF, blood was removed from the heart of mice using challenge with OVA. a syringe for measurement of OVA-specific IgG1, IgG2a, and IgE. Serum levels of anti-OVA IgG1, IgG2a, and IgE were measured by ELISA. Ninety- Downloaded from Materials and Methods six–well plates were coated with either OVA or purified anti-IgE (BD Mice Biosciences, Erembodegem, Belgium). After addition of serum samples, IgE standard (BD Biosciences), biotin rat anti-mouse IgE Ab (BD Biosciences), C57BL/6 mice were obtained from Harlan (Horst, The Netherlands). NTN2/2 goat anti-mouse IgG1 Ab, or goat-anti–mouse IgG2a Ab (SEROTEC, Ox- mice on C57BL/6 background were kindly provided by Prof. Jeffrey ford, UK) was used as detecting Ab, and the reaction was developed using Milbrandt (Washington University School of Medicine, St. Louis, MO). alkaline phosphatase. The OVA-specific Ab titer of the sample was related to

Mice were kept under specific pathogen-free conditions and were used at pooled standards that were generated in the laboratory and expressed as http://www.jimmunol.org/ 6–8 wk of age. All procedures respected current European regulations and ELISA units per milliliter. OVA-specific IgE concentrations were calculated were approved by the National Animal Research Authority. by comparison with the defined IgE standards. Sensitization and airway challenge Cytokine measurement Mice were sensitized with OVA (20 mg/injection; grade V; Sigma, Bornem, The levels of IL-4, IL-5, IL-6, IL-13, IL-17, IFN-g, and TNF-a in BALF, Belgium) emulsified in 2.25 mg aluminum hydroxide (Sigma) by i.p. lung, and lung draining LN culture supernatants were measured by injections in a total volume of 200 ml PBS on days 1, 14, and 21, followed Cytometric Bead Array (CBA; BD Biosciences), according to the manu- by 1% OVA in 50 ml normal saline administered intranasally after a short facturer’s protocol. anesthesia with halothane on days 27, 28, and 29. Control mice received i. Lung histology p. injections of aluminum hydroxide in PBS and were challenged with by guest on September 28, 2021 normal saline intranasally. On day 30, AHR was measured as described After BALF, the lungs were collected, fixed in 4% paraformaldehyde, and later, followed by collection of samples for further analysis. embedded in paraffin. Lung tissue sections were stained with H&E or with Intranasal NTN treatment of mice periodic acid-Schiff (PAS) to assess the airway inflammation and goblet cell metaplasia, respectively. All slides were read by an observer who was After a short anesthesia with halothane, 50 ml(5mg/ml) recombinant blinded to the treatment groups. The extent of peribronchial inflammatory mouse NTN (R&D Systems, Abingdon, U.K.) diluted in PBS was ad- cell infiltration around the bronchi in the lung tissue was quantified on a 0–3 ministered intranasally 1 h before the OVA challenge on days 27, 28, and scale. The histology in untreated control mice was defined as 0, and max- 29. Control mice received PBS 1 h before the OVA or PBS challenge on imum changes were given a score of 3 as previously described (16). Pictures days 27, 28, and 29. were taken with a Leica microscope with a camera (model DMI 6000B). Determination of airway responsiveness RT-PCR Airway function was assessed by measuring changes in lung resistance and Total RNA was extracted from lung, brain, and lung draining LN tissues dynamic compliance (Cdyn) in response to increasing doses of inhaled with the RNeasy mini (Qiagen, Venlo, The Netherlands). cDNA was

FIGURE 2. OVA-specific Abs in the serum. Serum titers for OVA-specific Ab in NTN2/2 and WT mice were determined after sensitization and challenge. Data are derived from three mice per group of five independent experiments (n = 15). The results for each of the groups are expressed as means 6 SEM. Significant differences (*p , 0.05, ***p , 0.001) between the WT OVAversus NTN2/2 OVA and PBS versus OVA groups are observed. EU, ELISA units. The Journal of Immunology 6499 Downloaded from FIGURE 3. A, Cellular composition of BALF. Mice were sensitized and challenged as described in Materials and Methods. Data are derived from three mice per group of four independent experiments (n = 12). The results for each group are expressed as means 6 SEM. There are significant differences (**p , 0.01) between the groups WT OVA versus NTN2/2 OVA. B, Cellular composition of isolated lung cells. Lung cells were prepared from sensitized and challenged mice. The results for each group (n = 12) are expressed as means 6 SEM. The differences (**p , 0.01) between the groups WT OVA versus NTN2/2 OVA are significant.

reverse transcribed from 1 mg total RNA by using Primescript Reverse 780 (eBioscience, San Diego, CA). The analysis was performed as de- http://www.jimmunol.org/ Transcriptase (TAKARA, Shiga, Japan), according to the manufacturer’s scribed previously (17, 18). instructions. For the PCR experiment, the TAKARATaq was used. Primers for GFRa-2 were as follows: forward, 59-CTC TGG TGG GCT CCC AAG Statistical analysis 9 9 9 CTT GCC-3 ; reverse, 5 -GAG TCC CCG GCC ACA GCC CCA TC-3 ; The statistical significance of differences was determined by one-way 9 size, 328 bp. Primers for GAPDH were as follows: forward, 5 -TGA GTA ANOVA Newman–Keuls test. The values were expressed as means 6 9 9 TGT CGT GGA GTC TAC-3 ; reverse: 5 -AGT TGT CAT ATT TCT CGT SEM from independent experiments. Any difference with a p value ,0.05 9 GGT TC-3 ; size, 200 bp. PCR synthesis was run for 35 cycles (2 min at was considered significant. 94˚C, 30 s at 94˚C, 30 s at 65˚C for GFRa-2 and 58˚C for GAPDH, 1 min at 72˚C) followed by a final extension at 72˚C for 7 min. Results a by guest on September 28, 2021 Flow cytometry NTN receptor GFR -2 mRNA expression in lung and lung draining LNs Anti-mouse Ab used for flow cytometry were as follows: CD19-FITC and Gr-1–PE (Immunotools, Friesoythe, Germany), CCR3-PE (R&D Systems), The presence of GFRa-2, the NTN receptor, has been observed in NK1.1-PECy7, CD11c-allophycocyanin, and CD3-allophycocyanin-Alexa mouse lung by Northern blot analyses (19). We have corroborated

FIGURE 4. Lung histology. Representative sections of bronchi of WT (A, C, E, G) and NTN2/2 mice (B, D, F, H), not treated (A, B, E, F)orOVA- sensitized and challenged (C, D, G, H). Sections were stained with H&E (A–D) or PAS (E–H). Data shown represent one of four experiments with three mice in each group. Scale bars, 100 mm. I, Peribronchial infiltration and mucous secretion score measured as described in Materials and Methods. The differences (***p , 0.001) between the groups WT OVA versus NTN2/2 OVA are significant. 6500 NEURTURIN INVOLVED IN ALLERGIC ASTHMA this observation by RT-PCR: the expected GFRa-2 transcripts Furthermore, we observed significantly reduced IgG1 levels and were detected in lung tissue and in lung draining LNs (Fig. 1). decreased IgG2a levels in OVA-sensitized NTN2/2 mice. This finding indicates that the gene of the NTN receptor is tran- Eosinophil accumulation in BALF and lung scribed in tissues localized at the site of inflammation in our an- 2/2 imal model. Sensitization and challenge with OVA in NTN and WT mice affected the composition of the cells recovered in BALF and lung Ab responses to OVA sensitization and challenge tissue. In both groups of negative control mice, macrophages were To investigate the role of NTN in allergic airway inflammation, the predominant cell type in BALF (Fig. 3A). However, after we used OVA-sensitized NTN2/2 and wild-type (WT) mice. One sensitization and challenge with OVA, the predominant cells in the hallmark of allergic asthma is the production of allergen-specific BALF and in the lung were eosinophils in both NTN2/2 and WT IgE. As shown in Fig. 2, in nonsensitized mice (PBS i.p.), anti- mice. In NTN2/2 mice, a significantly higher eosinophil accu- OVA Ab could not be detected in serum. After sensitization and mulation was observed in the BALF (43 6 3.9% SEM) compared challenge with OVA, the IgE levels were significantly higher in with WT mice (29 6 3.6% SEM) (Fig. 3A). The cellular com- immunized NTN2/2 mice compared with their WT counterparts. position of lung tissues also showed a marked increase of Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 5. A, Cytokine levels in BALF of WT and NTN2/2 mice. IL-4, IL-5, IL-6, IL-13, IL-17, TNF-a, and IFN-g were measured in supernatants by CBA. Data are derived from three mice per group of four independent experiments (n = 12). The results for each group are expressed as means 6 SEM. Significant differences (*p , 0.05, **p , 0.01, ***p , 0.001) between the groups WT OVA and NTN2/2 OVA versus negative control and WT OVA versus NTN2/2 OVA. B, Cytokine levels in lungs of WT and NTN2/2 mice. The results for each group (n = 12) are expressed as means 6 SEM. Significant differences (*p , 0.05, **p , 0.01, ***p , 0.001) between the groups WT OVA and NTN2/2 OVAversus negative control and WT OVAversus NTN2/2 OVA. C, Cytokine levels in lung draining LN of WT and NTN2/2 mice. IL-4, IL-5, and IL-13 levels were measured in supernatants by CBA. The lung draining LN cells were restimulated in vitro with IL-2 or OVA for 3 d. Data are derived from three mice per group of three independent experiments (n = 9). The results for each group are expressed as means 6 SEM. Significant differences (***p , 0.001) between the groups WT OVA versus NTN2/2 OVA. The Journal of Immunology 6501 eosinophils (Fig. 3B). Similar to the BALF, there was a signifi- The cytokine data from BALF, lung tissue, and lung draining LN cantly higher level of eosinophils in lung tissue of NTN2/2 of immunized animals demonstrate a consistent pattern of much compared with WT mice (20.5 6 2.3% SEM and 14 6 2.3% higher Th2 cytokine levels (IL-4, IL-5, and IL-13) in NTN2/2 SEM, respectively) (Fig. 3B). compared with WT mice, whereas levels of the other types of cytokines (Th1 and Th17) were not significantly affected by the Pulmonary inflammation absence of functional NTN genes. Lung histology was assessed 24 h after the last OVA challenge. In Airway responsiveness control groups, H&E staining revealed no airway inflammation (Fig. 4A,4B). In contrast, a significant eosinophilic and mono- We then investigated whether a deficiency of NTN could influence nuclear cell infiltrate was apparent in both perivascular and peri- AHR. AHR was measured in response to methacholine, 1 d after 2/2 bronchial areas in OVA-sensitized WT mice (Fig. 4C,4G). Under the last intranasal challenge with OVA or PBS in WT and NTN 2/2 the same conditions, sections from NTN2/2 lung tissue revealed mice. OVA-immunized WT and NTN mice showed signifi- that the level of infiltration of inflammatory cells into the peri- cantly increased pulmonary resistance in response to methacholine bronchial region was greater compared with those in WT mice (Fig. challenge compared with control mice (Fig. 6A). Furthermore, 2/2 4C,4D). The peribronchial infiltration scores were increased sig- OVA-immunized NTN mice displayed a significantly reduced 2/2 nificantly in NTN2/2 mice after OVA sensitization and challenge Cdyn compared with unimmunized NTN mice, indicating the compared with the score found in the WT mice (Fig. 4I). PAS successful induction of AHR in immunized mice (Fig. 6B). The 2/2 staining of mucus was negative in control groups (Fig. 4E,4F), AHR data of the resistance in immunized NTN mice were whereas mucous-producing epithelium was present after OVA significantly higher in comparison with their WT counterparts, Downloaded from challenge in WT and NTN2/2 mice (Fig. 4G,4H). In contrast with and the compliance data were significantly lower (Fig. 6A,6B). data obtained for cell infiltration, the mucous secretion score was Effects of NTN administration on cells and Th2 cytokine levels not higher in NTN2/2 mice compared with that seen in WT mice (Fig. 4G–I). The percentage of blood eosinophils was determined at To demonstrate directly the implication of NTN in eosinophil the time of tissue harvest. Eosinophil levels in immunized NTN2/2 recruitment and Th2 cytokine secretion after allergen challenge, we pretreated mice with NTN or PBS before the OVA challenge. We and WT mice were similar (n = 10, data not shown). http://www.jimmunol.org/ focused on cells and cytokines that had significantly different levels Cytokine levels in BALF and lung between immunized NTN2/2 and WT mice. The levels of eosi- To further investigate the effect of NTN on allergic inflammation, nophils, IL-4, IL-5, and IL-13 were analyzed in BALF, lung tissue, we measured cytokine production in the BALF and lung super- and lung draining LNs (Fig. 7A,7B). When NTN was adminis- tered before the OVA challenge, the percentage of eosinophils was natants by CBA. To observe all parameters implicated in in- 2/2 flammation processes, we investigated Th1, Th2, and Th17 path- significantly lower in BALF of WT and NTN mice compared ways through the level of TNF-a, IL-6, IFN-g, IL-4, IL-5, IL-13, and IL-17. The levels of IL-4 and IL-5, which play a crucial role in the recruitment of eosinophils, were significantly increased by guest on September 28, 2021 in OVA-sensitized WT and NTN2/2 mice compared with non- sensitized control mice (Fig. 5A,5B). In BALF and lung, IL-4 increase was significantly higher in immunized NTN2/2 mice compared with their WT counterparts (Fig. 5A,5B). IL-5 pro- duction was increased in immunized NTN2/2 mice, although this effect was not significant (Fig. 5A,5B). Furthermore, the level of the proinflammatory cytokine IL-6 was increased in NTN2/2 mice compared with WT mice after OVA sensitization and challenge. In contrast, BALF and lung supernatant content of IL-13 were sim- ilar in the immunized NTN2/2 and WT mice (Fig. 5A,5B). IL-17 and TNF-a are implicated in airway neutrophil recruitment (20). The level of these two cytokines did not vary between immunized NTN2/2 and WT mice (Fig. 5A,5B). There was also no difference for the IFN-g level, a cytokine implicated in macrophage re- cruitment and in Th1 pathway activation (21) (Fig. 5A,5B). Cytokines produced by lung draining LN cells in culture The lung draining LNs were also investigated because they are implicated in the inflammatory response, and the GFRa-2 receptor expression was detected in these organs. The ratio of CD4+ and CD8+ cells in the lung draining LNs did not differ between the OVA-sensitized WT and NTN2/2 mice (data not shown). The lung draining LN cells were isolated and restimulated in vitro with FIGURE 6. AHR. Lung resistance (RL)(A), and pulmonary Cdyn (B)in OVA for 1 and 3 d to investigate cytokine production. The increase sensitized and challenged mice. RL and Cdyn were obtained in response to increasing concentrations of methacholine (0, 6.25, 12.5, 25, and 50 mg/ of IL-4, IL-5, and also IL-13 production by OVA immunization 2/2 ml). Data are derived from three mice per group of three independent was significantly higher in NTN mice compared with WT experiments (n = 9). The results for each group are expressed as means 6 mice after 1 d (data not shown). This difference was even more SEM. Significant differences (*p , 0.05, ***p , 0.001) between PBS pronounced after 3 d of culture (Fig. 5C). In contrast, there were groups versus OVA groups are observed. Significant differences (#p , no significant differences in TNF-a, IFN-g, IL-6, and IL-17 pro- 0.05, ###p , 0.001) between the groups WT OVAversus NTN2/2 OVA are duction (data not shown). observed. 6502 NEURTURIN INVOLVED IN ALLERGIC ASTHMA with mice receiving only OVA challenge. The same tendency was cumulation and activation in bronchial tissues contribute to airway observed in the lung (Fig. 7A). Furthermore, the level of IL-4 was remodeling in chronic asthma (22). By using NTN2/2 mice, we significantly lower in BALF of WT mice and NTN2/2 mice after clearly showed that the absence of NTN is linked to a significant NTN administration (Fig. 7A). The same phenomenon was ob- increase in eosinophilic infiltration in BALF and lung tissue. served for the level of IL-4 in WT lung tissue. Adding NTN in- Histological analyses confirm that the absence of NTN increases duced a slight decrease of IL-5 in BALF of OVA-sensitized WT the peribronchial inflammatory cell infiltration in the lung tissue. and NTN2/2 mice, and a significantly weaker level of IL-5 was NTN apparently has no influence on mucus development. Ac- observed in WT lung tissues (Fig. 7A). The levels of IL-4 and IL-5 cording to our data, the increase of eosinophils in the peribron- in lung draining LNs in presence or absence of NTN did not vary chial regions, BALF, and lung tissues in NTN2/2 mice was not a lot, although the tendency was a decrease of IL-5 level when correlated to an increase of blood eosinophils. NTN was administrated (Fig. 7B). Lastly, IL-13 was also signifi- The Th2 cytokines IL-4, IL-5, and IL-13 are known to be in- cantly lower in lung draining LNs from NTN2/2 mice challenged creased in asthmatic BALF, lungs, and lung draining LN cells, and with OVA and NTN compared with NTN2/2 mice only chal- play major roles in the development and maintenance of the lenged with OVA. These results demonstrated that adding NTN in asthmatic IgE-driven conditions (23–25). In our study, the lack of our mouse model of airway inflammation partially rescues the NTN expression in OVA-sensitized mice led to strikingly in- NTN2/2 phenotype. Our data identify the importance of NTN as creased levels of IL-4, IL-5, and IL-13. The increased Th2 cyto- an anti-inflammatory molecule in the development of allergic kine levels were accompanied by significantly increased amounts airway inflammation. of OVA-specific IgE Ab. TNF-a and IL-17, which are implicated 2 2 in the recruitment of neutrophils (20), were not affected in NTN / Downloaded from Discussion mice compared with WT mice. At the same time, the level of Asthmatic airway inflammation and AHR development are or- neutrophils also did not differ between NTN2/2 and WT mice. chestrated by different cell types and mediators. Eosinophil ac- IFN-g, which is thought to counterbalance Th2 cytokine effects http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 7. Effects of NTN administration. Mice were sensitized and challenged as described in Materials and Methods. Data are derived from three mice per group of two independent experiments (n = 6). A, Eosinophil, IL-4, and IL-5 levels in BALF and lung tissues. Results for each group are expressed as means 6 SEM. Significant differences (*p , 0.05, **p , 0.01, ***p , 0.001) between OVA groups versus OVA+NTN groups are observed. B, IL-4, IL- 5, and IL-13 cytokine levels in lung draining LNs were measured in supernatants. The lung draining LN cells were restimulated in vitro with IL-2 or OVA for 3 d. Results for each group are expressed as means 6 SEM. Significant differences (*p , 0.05) between OVA groups versus OVA+NTN groups are observed. The Journal of Immunology 6503

(21), was not significantly decreased. These data demonstrate that NTN is expressed by immune cells and delivers an anti- a Th2 shift in NTN2/2 mice and show that, in our model, Th1 and inflammatory message that reduces migration or survival of Th17 pathways were not affected by NTN. eosinophils. Indeed, NTN is a molecule that influences cell pro- A characteristic feature of chronic asthma is AHR. Mediators liferation and differentiation in the case of neurons (34). In con- secreted during inflammation contribute to the development of clusion, our findings provide good evidence that allergic airway AHR. IL-4, IL-5, and IL-13 have the capacity to influence airway inflammation is regulated, at least in part, by NTN. These smooth muscle contraction and to induce AHR (26, 27). In ad- observations suggest that NTN and its receptor could become dition, IgE-mediated cytokine production by mast cells may potential therapeutic targets in asthma. contribute to AHR (28). Our data show that the lack of NTN significantly increases the OVA-induced AHR to inhaled meth- Disclosures acholine. This may be associated with the increase in Th2 cyto- The authors have no financial conflicts of interest. kine production, airway eosinophilia, and specific IgE levels. Indeed, the absence of NTN significantly increased these param- eters in OVA-immunized mice. References When NTN was administrated in our mouse model of in- 1. Robinson, D. S., Q. Hamid, S. Ying, A. Tsicopoulos, J. Barkans, A. M. Bentley, 2 2 flammation, we partially rescued the NTN / inflammatory C. Corrigan, S. R. Durham, and A. B. Kay. 1992. Predominant TH2-like bron- phenotype. Eosinophil and Th2 cytokine levels were significantly choalveolar T-lymphocyte population in atopic asthma. N. Engl. J. Med. 326: 298–304. decreased in the presence of NTN. This neurotrophin was able to 2. Nassenstein, C., J. Kutschker, D. Tumes, and A. Braun. 2006. 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