Marmoset monkeys (Callithrix jacchus) develop eosinophilic airway inflammation after house dust mite exposure

Background Extensive analysis of eosinophilic airway inflammation in human-relevant animal models is required to test novel, human-specific pharmaceuticals. This requires species, which show high genetic homology to humans such as non-human primates. Efficacy assessment of novel human-specific biologicals in eosinophilic airway inflammation is currently performed in the cost-intensive macaque asthma model. Objective The present study investigated whether marmoset monkeys (Callithrix jacchus), a small-bodied non-human primate species from the New World, develop eosinophilic airway inflammation in response to house dust mite allergen exposure (HDM, Dermatophagoides pteronyssinus). Methods Marmoset monkeys were sensitized against HDM by subcutaneous (s.c.) injection and subsequent intratracheal (i.t.) HDM aerosol challenges. Airway and systemic immunologic reactions were monitored and sensitivity towards glucocorticoid therapy was assessed. The pulmonary immunologic response was analyzed by repetitive bronchoalveolar lavage (BAL). Results Bronchoalveolar lavage fluid (BALF) exhibited increased levels of eosinophils, mast cells, and lymphocytes, as well as interleukin (IL)-13 after HDM challenges, compared to negative controls. The systemic immunologic response was assessed in peripheral blood mononuclear cells derived from sensitized animals, which secreted increased IL-13 and IFN-γ upon allergen stimulation in contrast to non-sensitized negative control animals. Although IgE was not detectable, HDM-specific serum IgG was elevated in sensitized animals. Both airway and systemic responses were reduced by treatment with glucocorticoids. However, lung function and pathological analyses did not reveal significant differences between groups. Conclusion In conclusion, marmoset monkeys developed a mild HDM-induced eosinophilic airway inflammation useful for efficacy testing of novel human-specific biologicals.


INTRODUCTION 54
About 235 million patients worldwide suffer from eosinophilic allergic inflammation like asthma and 55 require therapeutic interventions [1]. Current therapeutics comprise glucocorticoids and long-acting beta-56 agonists, which in most cases efficiently control mild and moderate asthma. Severe asthma, however, is 57 frequently therapy-resistant, and only a few monoclonal antibodies such as omalizumab, mepolizumab, and 58 reslizumab have been approved for treatment [2]. The preclinical development of monoclonal antibodies 59 and other human-specific biologicals directed against a particular target requires high homology in rodents.

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If cross-reactivity is not given, non-human primates (NHPs) are often the species of choice.

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To analyze the efficacy of human-specific therapeutics in NHP models of asthma, symptoms and pathology 63 have to coincide with human asthma patients. In patients, asthma is characterized by localized airway 64 reactions, an increase of inflammatory markers, and airway obstruction as a result of pathological changes 65 [3]. Sensitization is triggered by inhaled allergens like house dust mite allergen (HDM), leading to an influx 66 of inflammatory cells into the airways. Inflammatory cells, which typically increase in airways of asthmatic 67 patients, include eosinophils and lymphocytes (reviewed in [4]), accompanied by the T-helper 2 (Th2) 68 cytokines IL-4, IL-5, and IL-13. Systemic markers of asthma, which can be detected in blood, include 69 allergen-specific IgE and Th2-derived interleukins (reviewed in [5]). Airway obstruction is determined by 70 lung function analysis and characterized by an early airway response (EAR) immediately after allergen 71 exposure and increased airway hyperresponsiveness (AHR) towards unspecific stimuli such as 72 methacholine [6]. As an alternative to in vivo lung function, these changes can also be observed in ex vivo 73 precision-cut lung slices (PCLS) [7]. Underlying inflammation-induced chronic pathological changes are 74 characterized by goblet cell metaplasia, subepithelial fibrosis, smooth muscle hypertrophy, and angiogenesis 75 [8].

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Additionally, a reduction of mast cells (7.3 x 10 2 cells/ml vs. 5.4 x 10 2 cells/ml) and lymphocytes (1 x 10 4 256 cells/ml vs. 7.1 x 10 3 cells/ml) were observed in budesonide-treated animals ( Fig. 2D   showed a significant increase of IL-13 (67.1 pg/ml) and IFN-γ (29.3 pg/ml) 96 h after HDM stimulation 289 compared to medium controls ( Fig. 4A and B). It was possible to attenuate these effects by adding the 290 glucocorticoid dexamethasone to the cultures ( Fig. 4A and B Figure S2), which may link 338 sensitization to respiratory inflammation. Besides the increase of antibody titers, the humoral response 339 showed an increase of CRP in allergic animals (Supplementary Figure S3). As an acute-phase protein, CRP 340 is generally induced by IL-6 and has been reported to be increased in allergic patients [34]. CRP is indicative 341 of early systemic pro-inflammatory response in humans as well as marmosets.

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The protocol for sensitization used in our study did not lead to impairment of in vivo lung function.