A new house dust mite–driven and mast cell–activated model of asthma in the guinea pig

Animal models are extensively used to study underlying mechanisms in asthma. Guinea pigs share anatomical, pharmacological and physiological features with human airways and may enable the development of a pre‐clinical in vivo model that closely resembles asthma.


| INTRODUC TI ON
Asthma is a heterogeneous disorder of the airways characterized by variable airflow obstruction and airway hyperresponsiveness (AHR). In allergic asthma, the exposure to innocuous airborne antigens leads to respiratory symptoms, including wheeze, cough, chest tightness and breathlessness as a consequence of excessive airway narrowing. 1,2 This allergen-induced bronchoconstriction is largely mediated by mast cells, which release a broad range of preformed and de novo synthetized mediators that lead to airflow obstruction. [3][4][5] The continuous exposure to allergens results in chronic inflammation and airway remodelling that further compromise airway function, though the mechanisms for the development of these features are still largely undefined. Access to a physiologically relevant in vivo model for mechanistic research in this complex disease involving a systemic reaction and alterations of lung function is essential for improved disease understanding and the development of novel therapies. Today, these investigations are dominated by mouse models of asthma. However, for studying functional lung responses, mouse airway smooth muscle (ASM) has limitations in that it does not respond to several important agonists such as histamine and cysteinyl-leukotrienes (Cys-LTs) 6 and also do not show antigen-induced bronchoconstriction (AIB) since very few mast cells are located in the peripheral parts of the mouse lung. 7 Moreover, murine airways also lack the bronchial circulation, mucus glands and the inhibitory innervation of their ASM that counteract the acute asthmatic attacks. 6 Therefore, to investigate the mechanisms that drive airway bronchoconstriction, there is a need for new improved pre-clinical models that asthmatic features in humans.
The guinea pig has been widely used for asthma modelling because they share several resemblances with humans in lung anatomy, pharmacology and physiology. As in allergic asthma, guinea pig airways display a robust initial response to histamine and leukotrienes, which are largely released by mast cells to induce bronchoconstriction. 8 Mast cell activation also leads to release of cytokines, chemokines and growth factors, 9 which influence inflammatory and immunological reactions, as well as remodelling.
As mast cells are distributed throughout the lung, mast cells interact with structural and immune cells along the whole airway tree.
This emphasizes the value of measuring the functional responses in both the proximal and distal parts of the lung, especially as it is emerging that alterations of the peripheral part of the lung are important in asthma. 10,11 Previous research in guinea pig asthma models has not studied the functional differences between different lung compartments, and there is a need to include the use of forced oscillation technique (FOT) for more accurate investigations of lung function measurement.
Although the OVA-driven model of asthma has been extensively applied in guinea pigs, OVA is not a trigger of human asthma. 6 Instead, current asthma models in animals are moving towards using more clinically relevant allergens, particularly those from the HDM, 12 as it has proven to induce a multifaceted immune response involving both the innate and adaptive arms of the immune system that do not appear in OVA models in mice. We have therefore developed a protocol using intranasal administration of HDM that may be used to investigate the AIB and AHR by FOT as well as to follow the early allergic reactions with whole-body plethysmograph during the whole time period. Here, we describe an asthma-like model in the guinea pig that reproduces the hallmark features of allergic asthma such as allergen-induced bronchoconstriction (AIB), AHR and type 2 inflammation with increased numbers of eosinophils and mast cells, and airway remodelling.

| Animals
Male Dunkin-Hartley guinea pigs were obtained from Envigo (Horst, The Netherlands) and housed on 12/12h light/dark cycles with food and water ad libitum. Following an acclimation period, animals with body weight range of 500-600g were used for experiments. All experiments were approved by the Stockholm ethics committee for animal research (permit number N143-14).

| Protocols of sensitization and challenge to HDM
Lyophilized Dermatophagoides pteronyssinus HDM extract was obtained from Greer Laboratories (Lenoir, NC, USA) and re-suspended in sterile PBS at a concentration of 2.1 mg/mL, and aliquots were stored at −80°C. All experiments were performed using the same HDM batch. Guinea pigs were sensitized to HDM through intranasal route on day 1 (0.5 mg/mL) followed by a booster dose given on day 4 (1.0 mg/mL). Beginning on day 15, animals were intranasally challenged with HDM (0.25 mg/mL) once per week for five consecutive weeks. All instillations were conducted in a volume of 100 µL. The timing and concentrations were based on unpublished experiments and experience with OVA-driven guinea pig models. 13 Animals were lightly anaesthetized with 5% isoflurane (Baxter, Deerfield, Illinois, USA) mixed in oxygen followed by a maintenance dose of 2% isoflurane prior intranasal instillations. The immediate airway response induced by HDM was investigated after the first challenge at day 15 ( Figure 1A), whereas airway responsiveness, inflammation and remodelling were measured after all five challenges at day 44. Agematched PBS-treated guinea pigs were used as controls.
in guinea pigs. This model may be suitable for mechanistic investigations of asthma, including evaluation of the role of mast cells.

| Assessment of HDM-induced bronchoconstriction by invasive forced oscillatory technique
On day 15, guinea pigs were anaesthetized intraperitoneally with 40 mg/kg ketamine (Ketalar, Pfizer, Sandwich, UK) and 5 mg/kg xylazine (Bayer, Leverkusen, Germany), and the trachea was exposed and cannulated. Heart rate and oxygenation were monitored throughout the experiment, and the body temperature was maintained at 37°C with a heating pad. Animals were connected to a computer-controlled ventilator, equipped with a module 4 (flexiVent FX, SCIREQ Inc, Montreal, Qc, Canada), and ventilated at respiratory rate of 60 breaths per minute and tidal volume of 10 mL/kg with a positive end-expiratory pressure (PEEP) of 3 cmH 2 O. Following 10 minutes of regular ventilation for stabilization, two baseline recordings of respiratory mechanics were obtained. Guinea pigs were initially exposed to PBS over 90 seconds followed by 3-minute measurements, and after that exposed to either HDM (0.25 mg/mL) or PBS over 90 seconds. Intratracheal challenge was by nebulization (Aerogen Ltd, Galway, Ireland), and respiratory mechanics was assessed for 30 minutes. To investigate the non-allergic effect of HDM on the bronchoconstriction, an additional group of non-sensitized (PBS) guinea pigs was exposed to HDM. All measurements were acquired using the flexiWare Software version 7.6 (Scireq Inc). Respiratory mechanics was analysed using the constant phase model to calculate the (Newtonian resistance R n ), tissue damping (G) and tissue elastance (H) parameters. An additional group of HDM-sensitized guinea pigs was treated with a combination of the selective hista-

| Assessment of HDM-induced changes in respiratory pattern by non-invasive barometric plethysmography
Non-invasive measurements of respiratory responses caused by intranasal challenges with HDM were performed using barometric Pressure signals inside the chamber were processed with eDacq Software version 1.8 (EMMS), and respiratory parameters, including the enhanced pause (Penh), were acquired. Penh was obtained according to principles previously described [14][15][16] and expressed as percentage increase over baseline values.

| Assessment of airway responsiveness to methacholine
Respiratory mechanics were assessed 24 hours after the fifth challenge using forced oscillation technique. Guinea pigs were anaesthetized with ketamine and xylazine, and additional doses were administrated as needed. Animals were placed on a warm pad, and the trachea was exposed and connected via a cannula to flexiVent FX as described above. Following standard lung volume history, two baseline measurements were acquired. Animals were then exposed to PBS aerosols for 10 seconds followed by respiratory measurements. Increased doses of methacholine (Sigma-Aldrich, St. Louis, MO, USA) (0.018-0.32 mg/mL) were given at 7-minute intervals.
Because guinea pig airways are extremely reactive to contractile agonists, low doses of methacholine were chosen based on previous studies. 17,18 R n was considered to calculate the provocative dose 200% (PD 200 ), that is the interpolated methacholine dose that caused a threefold increase of basal value.

| Cell counts in bronchoalveolar lavage fluid
Following lung function measurements, guinea pigs were killed with an overdose of ketamine and xylazine, and blood was collected by cardiac puncture. Lungs were flushed two times with 5 mL of sterile saline (0.9% NaCl), and the recovered bronchoalveolar lavage fluid (BALF) was pooled. BALF was centrifuged and separated into cellfree BALF for measurement of inflammatory mediators and BALF cells for cell counting and cytospins. Total cells were counted using a Bürker haemocytometer, and ≤50 000 cells were used for cytospins.
Cells were stained with May-Grünwald-Giemsa (Histolab Products AB, Gothenburg, Sweden) according to the manufacturer's protocol.
Leucocytes were identified based on morphologic criteria and quantified by counting 300 cells per slide.

| Cytokine analysis
Cytokines were quantified in BALF by sandwich ELISAs (Nordic BioSite; Täby Sweden) according to the manufacturer's protocol.

| Quantification of serum HDM-specific immunoglobulin levels
HDM-specific immunoglobulins were measured using the antigencapture ELISA method according to standard protocols. Anti-guinea pig IgE (Nordic BioSite AB, Täby, Sweden), and anti-guinea pig IgG 1 (246-GAGp/IgG1/Bio) and IgG 2 (246-GAGp/IgG2/Bio) (both from Nordic Immunological Laboratories, Susteren, The Netherlands) were used for the detection of immunoglobulin subclasses. Diluted serum samples were added to wells with or without capturing antigen, and the delta absorbance at 490 nm (ΔABS 490 nm) was calculated as the difference between wells with or without antigen capture. Caudal right lobe sections were stained with Astra blue (Sigma-Aldrich), which is a cationic dye that binds specifically to heparin contained in mast cell granules. 19 Stained airways were randomly selected under low power light microscopy (x40 magnification) and captured by using an Olympus UC50 camera (Olympus Australia, Melbourne, VIC, Australia) attached to an Olympus

| Statistical analysis
All data were analysed using GraphPad Prism 8 Software (GraphPad Software Inc, La Jolla, CA, USA) and are presented as the mean ± SEM.
Statistical analysis was performed using one-way or two-way ANOVA followed by Dunnett or Tukey's multiple comparison test. In addition, unpaired Student's t test was included. Significant differences were defined as *P < .05, **P < .01 and ***P < .001.

| Mast cell mediators drive the HDM-induced bronchoconstriction in sensitized guinea pigs
To validate the effect of sensitization, we first investigated the respiratory mechanics in response to challenge with HDM using FOT ( Figure 1A). The animals were challenged with either PBS or HDM de- To approach the immunological response in the allergic reactions induced by HDM, allergen-specific immunoglobulins were quantified. HDM-treated guinea pigs showed a significant increase of HDM-specific IgG 1 and IgG 2 , but not IgE, as compared with the PBS-sensitized groups ( Figure 1C). Moreover, differential cell count of immune cells in BALF following the first challenge revealed that HDM induced a robust and significant increase in the number of eosinophils and lymphocytes in both HDM-and PBS-sensitized guinea pigs as compared with PBS-challenged animals ( Figure 1D). In contrast, HDM challenge induced a marked increase in the number of neutrophils only in PBS-sensitized guinea pigs ( Figure 1D). The number of macrophages was similar among the groups (data no shown).

| Repeated intranasal HDM challenges induce changes in the respiratory pattern of guinea pigs
To investigate the effect of prolonged exposure to HDM on the airway responses, we adopted a long-term protocol of repeated intranasal challenges applied for up to 5 weeks (Figure 2A). The

| HDM administration induces AHR in guinea pigs
To investigate whether the exposure to HDM affects the AHR, we assessed the airway responses to methacholine after five weeks of challenges using FOT. HDM-treated guinea pigs showed marked increase in R n compared with PBS-treated controls ( Figure 3A).
Moreover, HDM-challenged animals showed both higher G ( Figure 3B) and H ( Figure 3C) values than PBS-treated controls upon methacholine aerosols, indicating that HDM affects both conducting airways and tissue mechanics. In addition, HDM-treated animals displayed a marked decrease of methacholine PD 200 compared with PBS animals ( Figure 3D).

| Exposure to HDM promotes a marked increase of immunoglobulins and mast cell numbers in sensitized guinea pigs
We next examined whether repeated exposure to HDM through the respiratory mucosa alters the production of allergen-specific immunoglobulins and induces mast cell accumulation. HDM-treated guinea pigs exhibited increased levels of specific IgE, IgG 1 and IgG 2 as compared with PBS-treated controls ( Figure 4A). In addition, lung sections stained with Astra blue revealed a robust increase of mast cells around both proximal and distal airways in HDM-treated guinea pigs ( Figure 4B).

| Repeated intranasal HDM challenges result in airway inflammation
Analysis of cells in BALF revealed a significant induction of eosinophils in response to HDM challenge, but not of macrophages, neutrophils and lymphocytes ( Figure 5A). Histological examination of H&E-stained lung sections showed an extensive infiltration of inflammatory cells in HDM-challenged guinea pigs ( Figure 5B).

RAMOS-RAMÍREZ Et Al.
To investigate potential mediators that might play a role in the induction of allergic airway inflammation in this model, pro-inflammatory cytokines were quantified in BALF obtained 24 hours after the last challenge. HDM-treated guinea pigs exhibited significantly increased levels of the type 2 cytokine IL-13 but not IL-4 ( Figure 6A-B). Levels of the eosinophil chemotactic mediators, IL-5 and CCL11 (eotaxin-1), and the pro-inflammatory cytokine IL-6 in BALF were not significantly affected by repeated exposure to HDM ( Figure 6C-E).

| HDM challenge induces airway structural changes
Analysis of H&E-stained lung tissues revealed a significant increase of the airway subepithelial region of HDM-treated animals compared with PBS-treated controls ( Figure 7A). In addition, the ASM layer was slightly increased in HDM-treated animals but did not reach significance difference between the groups ( Figure 7A).   10,11 Another important similarity between human and guinea pig airways is that the acute response to allergen provocation is largely mediated by histamine and leukotrienes. 20,21 In agreement with this, we were able to abrogate the effects of HDM on lung mechanics through pharmacological Thus, the anatomic co-localization of mast cells and smooth muscle in the airway is an important factor to consider for accurate modelling of the allergen-induced bronchoconstriction that occurs in asthma.

| D ISCUSS I ON
To verify that HDM challenge induces allergic activation of mast cells, we quantified the levels of HDM-specific immunoglobulins following the first challenge and found that HDM-treated guinea pigs were indeed sensitized to the allergen as shown by increased levels of HDM-specific IgG 1 and IgG 2 compared with PBS-treated guinea pigs.
The induction of both IgG subclasses in response to allergens, including dust mites, has previously been described in guinea pigs. 18,[22][23][24] Furthermore, it has been shown that both immunoglobulins can induce the ASM contraction, 22,25 also in line with our study. In contrast, no differences in the HDM-specific IgE levels following the sensitization In the current study, we showed that the repeated intranasal challenge with HDM extract leads to AHR in guinea pigs. Although HDM was introduced for sensitization of guinea pigs already 1973 29 and found to elicit AIB after repeated intradermal injection 30 or five days of aerosol challenge, 31 no AHR to histamine or methacholine was found. 31 These observations suggest that HDM extract instilled directly into the airways likely induces a more intense mucosal-driven allergic response that causes AHR. Furthermore, as the responses were measured using FOT, the AHR was found in both central and peripheral airways. As this technique has not been used for airway responsiveness measurements before in guinea pigs, it opens for investigating new dimensions in the coming studies.
One potential driver of AHR in our model is the extensive air- with asthma, and they have been recognized as markers of lung remodelling. 32,33 In guinea pig OVA models, this has been shown after 15 days or after 13 weeks of OVA exposure. 34 In addition, we observed an increase in the density of ASM upon repeated HDM exposures, although this did not reach statistical significance. The thickness of ASM layer is the major abnormality that affects the airway physiology in asthmatic subjects. 35 In many animal models, high doses of allergen are used to achieve structural changes in the airways. However, prolonged exposure to high doses of allergen, particularly in OVA-driven models, may induce the development of tolerance, 36 whereas low doses of allergen may result in too mild allergic responses. [37][38][39] In our study, the allergen tolerance was avoided by using increasing doses of HDM during the sensitization phase followed by several challenges with low HDM doses sufficient to cause airway remodelling.
Evaluation of the airway inflammation, which also is implicated in In addition, human recombinant IL-13, but not IL-4, can induce the differentiation of guinea pig tracheal epithelial cells into mucus-producing goblet cells. 42 Consistent with this, the repeated HDM challenge led to up-regulation of IL-13, but not other type 2 mediators such as IL-4 and IL-5. Notably, airway eosinophilia was not associated with the IL-5 and CCL11 production. However, this likely was not the optimal time point to detect elevated levels of cytokines as eosinophilic inflammation was already established in the airways.
Although the underlying mechanisms of AHR remain poorly understood, the induction of IgG 1 sensitization has been related to a greater contractile response of ASM in the guinea pig. 13,43 In our study, intranasal HDM administration induced high levels of allergen-specific IgG 1 and IgG 2 in both the short-and long-term protocols and increased allergen-specific IgE level following the repeated challenges. In agreement with our findings, Nabe and colleagues found that repeated inhalations of OVA induced an IgG 1 responses already at an early phase of the protocol, whereas IgE appeared only after repeated challenges. 44 Thus, in our HDM-driven model, multiple doses of the allergen over time might be required to induce IgEmediated responses, whereas IgG 1 -and IgG 2 -mediated responses are activated by low levels of HDM. As mast cells from guinea pig sensitized with IgG 1 release higher levels of histamine than those sensitized with IgE, IgG 1 has frequently been associated with allergic responses in the guinea pig. 22,45,46 These data suggest that mast cells may drive the airway dysfunction in our HDM-driven asthma model in guinea pig. Given that mast cells play a key role in the pathobiology of asthma, one of the major advantages of using guinea pigs for asthma studies is that distribution and function of mast cells is similar to that in human. 7,47 In our model, mast cell-dependent reactions, AHR and airway inflammation, were achieved upon the repeated intranasal challenge.
Taken together, the purpose of this paper was to establish a new asthma model by the use of a species that physiologically, anatomically and pharmacologically has great similarities to humans 6 using a method for sensitization with a relevant allergen through respiratory mucosa that activates both the adaptive and the innate immunity in the airways. The study provides evidence that the used protocol generates several key features of asthma such as AIB, AHR, remodelling and recruitment of inflammatory cells (including mast cells) together with an increase of IL-13. The model is therefore clearly relevant to human asthma and provides new opportunities to investigate the immunological processes and the mechanisms behind AHR, remodelling and recruitment of inflammatory cells. With this first step in this direction, we wish to demonstrate a model that can be used for future opportunities to study specific cell types and/or mediators, including the involvement of mast cells in asthma.

ACK N OWLED G EM ENTS
Funding was provided by the Swedish Heart-Lung foundation,