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J. S. Hansen*, T. Alberg*, H. Rasmussen*, M. Lovik*| & U. C. Nygaard* .... J. S. Hansen et al. ...... phil accumulation in immune-mediated inflammation. Immunity.
BASIC IMMUNOLOGY doi: 10.1111/j.1365-3083.2011.02529.x ..................................................................................................................................................................

Determinants of Experimental Allergic Responses: Interactions Between Allergen Dose, Sex and Age J. S. Hansen*, T. Alberg*, H. Rasmussen*, M. Lovik*  & U. C. Nygaard*

Abstract *Norwegian Institute of Public Health, Oslo, Norway; and  Norwegian University of Science and Technology, Trondheim, Norway

Received 16 December 2010; Accepted in revised form 23 January 2011 Correspondence to: J. S. Hansen, Department of Environmental Immunology, Norwegian Institute of Public Health, P.O. Box 4404 Nydalen, NO0403 Oslo, Norway. E-mail: jitkahansen@ gmail.com

The prevalence of allergic diseases is influenced by sex and age. Although mouse models are widely used in allergy research, few experimental studies have examined the interaction effects of sex and age on allergy outcomes. Our aim was to investigate the individual and combined effects of sex and age on allergic sensitization and inflammation in two mouse models: an intraperitoneal (i.p.) and an intranasal (i.n.) sensitization model. We also investigated how the allergen immunization dose interacted with age and sex in the i.p. model. Female and male mice were immunized i.p. or i.n. with ovalbumin when 1, 6 or 20 weeks old. In both models, allergen challenges were performed by i.n. delivery. Serum antibodies, draining lymph node cytokine release and airway inflammatory responses were assessed. In the i.p. model, the antibody and cytokine levels and airway inflammation were highly influenced by immunization dose and age. The responses increased with age when using a low immunization dose, but decreased with age when using a high immunization dose. In the i.n. model, antibody production and airway tissue inflammation increased with age. Female compared with male mice generally developed more pronounced antibody and inflammatory responses. Relative to older mice, juvenile mice had augmented airway inflammation to allergen exposures. The study demonstrates that immunization dose, sex and age are highly influential on allergy outcomes. To better mimic different life stages of human allergic airway disease, murine models, therefore, require careful optimization.

Introduction Murine models investigating the mechanisms and potential treatments of allergic diseases are widely used [1]. In these models, allergic sensitization is achieved by allergen immunization via different routes to induce allergen-specific IgE production. Following airway challenges with the allergen, an inflammation dominated by eosinophils is established. Lower allergen doses generally lead to higher IgE production than higher doses [2]. Whether this applies to both male and female mice has not been described, as allergy studies most often are carried out in female animals. In adult humans, allergic diseases differ in severity and prevalence depending on the sex; women tend to be affected more severely and have a higher allergy and asthma prevalence compared with men [3, 4]. Therefore, an increasing number of studies address sexspecific problems related to allergy and asthma aetiology [3, 5–7]. Thus, experimental studies should include both sexes to better reflect the human situation.

In humans, it is further known that allergy and asthma prevalence in males and females differ depending on age; boys have higher disease prevalence compared with girls, but this is reversed after puberty [3, 8, 9]. It has rarely been considered how age influences the allergic immune response in experimental models. Generally, 6to 10-week-old mice are used, but an increasing number of studies investigate allergy in younger mice, particularly in relation to prenatal exposure [10–12]. As allergic diseases and asthma often occur in early childhood, research in developmental immunology requires specific experimental models to mimic this period of life. The effect of sex, to a lesser extent age, and very rarely a combination of these factors, has been addressed in experimental studies of allergy. Therefore, it was the aim of the presented studies to describe sex- and agerelated effects on allergy outcomes in two murine models. The age groups were selected to cover an age span that may be used in allergy models. In a first study, we investigated how the intraperitoneal (i.p.) immunization  2011 The Authors

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J. S. Hansen et al. Factors Affecting Experimental Allergy 555 ..................................................................................................................................................................

dose affected allergy outcomes after airway challenges in juveniles, adolescent and fully mature female and male mice. Such i.p. sensitization followed by airway challenges is widely used in experimental research. In a second study, a more realistic route of sensitization was used; female and male mice of the same age groups as used in the previous study were sensitized and challenged by intranasal (i.n.) allergen exposures only. In both models, allergen-specific antibodies in serum, cytokine release from airway-draining lymph nodes and airway inflammation were used as end points relevant for experimental allergy.

Materials and methods Mice. Age-matched inbred NIH ⁄ OlaHsd female and male mice (Harlan Ltd, Blackthorn, UK) were acclimatized for at least 2 weeks. For the 1-week-old groups, newborn mice were obtained for different experiments either by in-house mating or from time-mated females obtained from the breeder. To avoid litter effects, littermates were marked and allocated to different immunization groups. Offspring were weaned at 3 weeks of age and housed 2–3 mice per cage. Males more than 9 weeks old (or if necessary younger) were housed individually to avoid fighting. Mice were provided tap water and standard laboratory chow ad libitum. The mice were exposed to a 12-h light ⁄ dark cycle (30–60 lux in cages), regulated room temperature (20 ± 2 C) and 40–60% relative humidity. The experiments were approved by the Institutional Animal Care and Use Committee at the Norwegian Institute of Public Health and performed in conformity with the Norwegian laws and regulations and the European Convention guideline ETS123 on the care and use of experimental animals. Allergen, adjuvant and anaesthetics. Chicken egg ovalbumin (OVA), grade VII, was from Sigma-Aldrich, St. Louis, MO, USA. The Al(OH)3 adjuvant (Alhydrogel) was from Brenntag Biosector, Denmark. Two different types of anaesthetics were used; Isoflurane (Isoba vet; Intervet ⁄ Schering-Plough Animal Health, Lysaker, Norway) and a cocktail named ZRF, consisting of Zoletil Forte (Virbac International, Carros Cedex, France), Rompun (Bayer Animal Health GmbH, Leverkusen, Germany) and Leptanal (Janssen-Cilag International NV, Beerse, Belgium) and isotonic saline. Isoflurane gas was administered as a 3.5% mixture with medical O2 in a coaxially ventilated open mask to effect. The ZRF cocktail contains 18.7 mg Zolazepam, 18.7 mg Tiletamine, 0.45 mg Xylazine and 2.6 lg fentanyl per ml and was administered to effect with a nominal dose of 0.1 ml ⁄ 10 g i.p. Intraperitoneal sensitization study. Groups of mice received first sensitization at ages 1, 6 and 20 weeks and are hereafter referred to as 1-, 6- and 20-week-old mice. The mice were sensitized by i.p. administration of 0, 0.1,

10 or 1000 lg OVA in 1 mg Al(OH)3 in Hank’s balanced salt solution (HBSS) in a 0.1-ml bolus. Two weeks later, they were boosted i.p. with the corresponding dose, but without Al(OH)3 in 0.1 ml. All mice in the 1000lg groups suffered from severe anaphylactic chock and died or were killed upon booster administration. One week later, a blood sample was taken from the remaining groups, which were then anaesthetized with isoflurane and challenged by i.n. instillation of 10 lg OVA in 35 ll HBSS per day for 3 days. Three days after the last challenge, the mice were anaesthetized with ZRF before the chest was opened and blood drawn by heart puncture. Lung-draining mediastinal lymph nodes (MLNs) were collected, lungs lavaged and the lymph nodes and bronchoalveolar lavage fluid (BALF) kept on ice. Intranasal sensitization study. Groups of 1-, 6- and 20week-old mice were sensitized i.n. [13] with 10 lg OVA with 120 lg Al(OH)3 in HBSS on days 1, 2 and 3 (Table 1). On days 22, 23 and 24, they were boosted i.n. with 10 lg OVA in HBSS. All i.n. exposures were performed under isoflurane anaesthesia. On day 27, blood was drawn by heart puncture. Nose- and lung-draining lymph nodes [superficial cervical (SLNs) and MLNs, respectively [14]] were collected and kept on ice; lungs were lavaged and thereafter collected for histopathology. The BALF was also kept on ice. In a concurrent study, control groups of age- and sexmatched mice were immunized i.n. with OVA alone without Al(OH)3 (Table 1). This OVA-only exposure did not induce sensitization or any significant responses, when compared with OVA + Al(OH)3-treated mice. For clarity, the OVA-only groups are not presented, except for a few observations. Determination of instillation volumes in the intranasal sensitization study. The mice of the different age groups were exposed according to Table 1. For the adult mice, the choice of a 35-ll volume was based on a previous study determining this as the minimum volume for maximal lung deposition [15]. For use in mouse pups, optimal intranasal exposure volumes for lung deposition were determined. Volumes of 5-, 10- or 15-ll Evans Blue solution were deposited on the nostrils under isoflurane anaesthesia. After 15 min, the pups were killed by cervical dislocation. Table 1 Details of exposure in the intranasal sensitization study. Age group (week) Intranasal volume 1 6 20

10 30 35 35 35 35

ll ll ll ll ll ll

– – – – – –

sensitization challenge sensitization challenge sensitization challenge

Dose 3 3 3 3 3 3

· · · · · ·

10 10 10 10 10 10

OVA, ovalbumin.

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lg lg lg lg lg lg

OVA ± 120 lg Al(OH)3 OVA OVA ± 120 lg Al(OH)3 OVA OVA ± 120 lg Al(OH)3 OVA

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The 10-ll volume was determined to give maximal lung deposition by visual inspection of the blue-colouring of the lungs and stomach. Selection of 30 ll as the booster volume (Table 1) was based on the estimated 300% increase in body mass from 1 to 4 weeks of age [16, 17]. Determination of OVA-specific antibodies in serum. OVAspecific IgE antibodies were detected in a capture ELISA as previously described by Lovik et al. [18] and modified by Ormstad et al. [19]. Poly-HRP-streptavidin (Thermo-Scientific, Pierce Biotechnology Inc., Rockford, IL, USA) followed by Stabilized chromogen TBM (Invitrogen, Camarillo, CA, USA) was used for detection and the reaction stopped with 2 N H2SO4 solution. OVA-specific IgG1 was measured in a capture ELISA as previously described [13]. The sera to be tested were analysed in duplicates following optimal dilution; 1:50 for IgE and 1:200 or 1:200,000 for IgG1. For both antibody assays, a standard curve was included on each plate. Standard curves were made from duplicates of diluted IgE standard (mouse antiOVA IgE, AbD Serotec) or serum pools from mice immunized with OVA and Al(OH)3 for IgG1. OD was measured at 450 nm on a MRX Microplate Reader (Dynatech Laboratories, Chantilly, VA, USA) connected to a PC using Revelation software (Thermo Labsystems, Chantilly, VA, USA). Lymph node preparation and determination of cytokine release. Single-cell suspension of SLNs and MLNs was prepared by forcing the lymph nodes through a 70-lm cell strainer (BD Labware, Franklin Lakes, NJ, USA). The cells were washed and then counted in a Coulter Counter Z1. After incubation in culture medium (RPMI 1640 with 10% foetal calf serum, 100 U penicillin G, and 0.1 mg ⁄ ml streptomycin) with or without 1 mg ⁄ ml OVA at 37 C and 5% CO2 for 4 or 5 days (differed for practical reasons between the i.n. and i.p. study, respectively), the supernatants were removed and stored at )20 C until cytokine measurements. The levels of IL-4, IL-5, IL-10, IL-13, IFN-c, and in the i.n. study also IL-17, were determined using BD CBA Mouse Soluble Protein Flex Sets measured on a BD LSR II flowcytometer and analysed by the FCAP Array software (all from BD Biosciences, San Jose, CA, USA) according to the manufacturer’s protocol. Assessment of inflammatory cells and cytokines in BALF. Cells in BALF were prepared and stained with the Hemacolor rapid staining of blood smear kit (Merck KGaA, Darmstad, Germany) as previously described [13]. Cell differential counting of blinded slides was performed by microscopic examination by the same investigator (JSH). In the i.n. model, IL-5, IL-10, IL-17, RANTES, MIP-1a and MCP-1 in BALF (stored at )80 C) were measured with the BD CBA Mouse Soluble Protein Flex Sets as described earlier. Histopathology. The method was established after conduction of the i.p. sensitization study, thus applied only in the i.n. sensitization study. Following bronchoalveolar lavage, lungs were inflated and immersion-fixed in neutral

buffered formalin (10%), paraffin-sectioned at 5 lm thickness and stained with haematoxylin and eosin (H&E) or Periodic Acid Schiff (PAS). The inflammatory infiltrate and staining of goblet cells were evaluated by light microscopy of the H&E and PAS sections, respectively. All pathology scoring was performed by the same investigator (HR) that was aware of the animal grouping, but blinded to all other results. The intensity of the perivascular and peribronchial inflammatory infiltration was scored according to the following grading scheme. Lung sections graded as 0 showed no inflammatory infiltration. Sections graded as 1 demonstrated 1 or 2 minimal foci of perivascular and peribronchial infiltration, while grade 2 presented 3–6 foci of perivascular and peribronchial infiltration. Sections graded as 3 presented multiple foci of perivascular and peribronchial infiltration, many of which formed multilayered cuffs, while grade 4 presented multiple multilayered dense inflammatory infiltrates, primarily affecting the central parts of the lungs. Sections graded as 5 were as grade 4 but more extensive by affecting both central and peripheral parts of the lungs. Staining of goblet cells in the bronchi was graded as 0, 1, 2, 3 and 4, corresponding to PAS-positive staining of 5% or less, 5–15%, 15–30%, 30–50% and more than 50% of bronchial epithelial cells. A Zeiss Axioplan 2 microscope (Carl Zeiss, Go¨ttingen, Germany) with Plan-Neoflux 10 · ⁄ 0.30 lenses was used to magnify the histology slides. An RT Spot digital camera with the Spot RT slider v.4.6 software was used for image acquisition, addition and merger of electronic scale bar [using a Nikon MBM 11100 stage micrometre type A (Nikon, Tokyo, Japan) for objective calibration]. Adobe Photoshop CS4 v. 11.0 (Adobe Systems Inc., San Jose, CA, USA) was used for proportional resizing of the images. Image resampling during resizing was performed as bicubic sharper. Pixel order was interleaved (RGBRGB), and no compression was applied upon saving. Auto colour and auto contrast correction was applied to the entire image. No other adjustment of the images was performed. Study design and statistical analysis. A factorial design was used for both the i.p. and i.n. studies, which were analysed statistically by the General Linear Model procedure in Minitab v.15 (Minitab Inc., State College, PA, USA) with sex, age and allergen dose as fixed factors. When necessary, data were logarithmically or square root transformed to obtain equal variance and normal distribution of the residuals. Statistically significant main and interaction effect are reported. Post hoc testing by the Bonferroni method was performed when appropriate. Because the factor ‘age’ has three levels (1, 6 and 20 weeks), post hoc testing was performed in case of significant main effects of age. When significant interaction effects were found, these instead of significant main effects were evaluated statistically by post hoc analyses. Outcomes of post hoc tests are shown on the figures. For clarity, only significant and relevant comparisons are  2011 The Authors

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J. S. Hansen et al. Factors Affecting Experimental Allergy 557 .................................................................................................................................................................. Table 2 Statistically significant main and interaction effects in the intraperitoneal sensitization study. OVA-specific IgE Sex Dose Age Sex · Dose Sex · Age Dose · Age Sex · Dose · Age

0.003