Shortness of breath, wheezing, coughing, and chest tightness – these are the symptoms that affect 300 million people worldwide, with the reasons being obstructive lung diseases. Obstructive lung diseases do not only influence personal welfare, but they are also among the four most common causes of death worldwide, impairing both families and the economy (WHO, 2017). The collective term comprises the two diseases asthma and chronic obstructive pulmonary disease (COPD), which show increased prevalence at different stages in life. Whereas asthma is the most common chronic disease in children, the risk for COPD is increased later in life. To assess necessity of therapeutic intervention, both diseases are classified into different stages. The severity of asthma is classified according to the frequency of symptoms, lung function results, and requirements of therapeutics (National Asthma Education and Prevention Program, 2007). Similarly, COPD is categorized according to reduced lung function, the nature of the patient's symptoms, and the risk of exacerbation (GOLD, 2017).

Asthma symptoms occur due to airway obstruction either after an acute stimulus, e.g., an allergen, or as a result of chronic remodeling processes. In affected atopic patients, an acute asthmatic response occurs within minutes after allergen exposure, leading to reduced lung function due to airway obstruction, designated as early airway response (EAR). Mechanistically, allergens induce cross linking of mast cell bound immunoglobulin E (IgE), resulting in a release of mast cell mediators. These lead to airway obstruction by smooth muscle contraction, vasodilation, and increased vascular permeability. Several hours after this acute reaction, mast cell mediators induce an airway inflammation due to leukocyte influx in addition to mucus hypersecretion, which leads to another obstructive event, designated as late airway response (LAR; Galli and Tsai, 2012; Galli et al., 2008). Similarly, patients develop airway hyperresponsiveness (AHR) towards bronchial irritants, which is assessed in lung function analysis by methacholine exposure.

Repeated exposure to allergens leads to chronic inflammation, inducing structural remodeling processes in the airways. Inflammatory airway infiltrates are dominated by eosinophils accompanied by T lymphocytes, leading to epithelial cell injury. These cells are found in bronchoalveolar lavage (BAL) and biopsy material of asthmatic patients (reviewed in Howarth et al., 1994), associated with an increase in T helper (Th) 2 lymphocyte-derived immune mediators like the cytokines interleukin (IL)-4, IL-5, and IL-13 (Robinson et al., 1992; Huang et al., 1995; Kroegel et al., 1996). Remodeling processes include goblet cell metaplasia, subepithelial fibrosis, smooth muscle hypertrophy, and angiogenesis (Fahy, 2015), resulting in chronic airway obstruction.

Airway obstruction in COPD is a result of chronic exposure to irritants like smoke or air pollution. Continuous inhalation leads to inflammation of the airways, characterized by influx of primarily neutrophils; macrophages; and Th1, Th17, and cytotoxic T 1 (Tc1) lymphocytes. Whereas non-affected smokers develop only mild inflammation, smoking COPD patients develop an exaggerated inflammatory response, which remains after removing the irritant. The reason for this modified immune response is unknown, but genetic factors might contribute to disease outcome. Chronic bronchiolitis is accompanied by mucus hypersecretion and remodeling processes, including peribronchiolar and interstitial fibrosis resulting in airway obstruction. Chronic impairment of expiration, diagnosed in lung function analysis, leads to airway hyperinflation, thus inducing parenchymal destruction and emphysema formation (Barnes, 2016; GOLD, 2017).

Prevention of acute or chronically deteriorating symptoms requires ongoing therapeutic intervention both in asthma and COPD. Whereas mild and moderate asthma are well controlled by inhaled corticosteroids and long-acting beta-agonists (LABAs), severe asthma is largely therapy resistant. In these patients, alternative approaches involve application of therapeutic antibodies targeting IgE (omalizumab) and IL-5 (mepolizumab, reslizumab) (Brusselle and Bracke, 2014). Application of these antibodies significantly reduces exacerbations in severe asthmatics (Humbert et al., 2005; Ortega et al., 2014; Bel et al., 2014; Castro et al., 2015) but may not work if target antibody titers are above 3–4 % of total IgE (Johansson et al., 2009). Additionally, production costs are relatively high (Beck et al., 2010) and application is only approved in a clinical setting (EMA, 2009a) due to risk of anaphylactic reactions (reviewed in Khan, 2016).

Treatment of COPD patients requires ongoing treatment with long-acting muscarinic receptor antagonists (LAMAs) or LABA, which can be substituted with corticosteroids or other anti-inflammatory therapeutics, e.g., macrolides or phosphodiesterase-4 inhibitors. Whereas exacerbation can be reduced by inhaled glucocorticoids, they are not able to restore lung function capacity (Yang et al., 2012; Durham et al., 2016). Potential new targets include targeting the inflammasome, TNF-α (tumor necrosis factor alpha), IL-17 and IL-17 receptor, IL-18, and B cells (Brusselle and Bracke, 2014).

Overall, until today therapeutic approaches aim to prevent occurrence of symptoms and target structures which avoid amplification of existing inflammation. To prevent disease manifestation, causative mechanism of asthma and COPD need to be well understood in suitable animal models and human patients in translational approaches.

Ideal animal models for human diseases are those which naturally occur in an animal species, show identical pathology, and can be induced for direct availability. Obstructive lung diseases are mainly restricted to humans, whereas only a few animal species show naturally occurring diseases. Among these, horses and cats develop diseases similar to human asthma, and COPD-like symptoms appear in dogs (Williams and Roman, 2016). Nevertheless, recurrent airway obstruction in horses lacks eosinophilia and an acute airway response, while feline asthma shows comparable pathology to human asthma and is experimentally inducible (reviewed in Kirschvink and Reinhold, 2008). Dogs develop chronic bronchitis, while emphysema formation is only rarely observed (reviewed in Williams and Roman, 2016). Thus, among domestic animals the cat provides an attractive model for human asthma, but cross reactivity of human-specific biologicals might be absent for phylogenetic reasons.

Due to close genetic homology, nonhuman primates provide targets for development of human-specific therapeutics in obstructive airway diseases. Classically, the old world monkey species Macaca mulatta (rhesus macaques) and Macaca fascicularis (cynomolgus macaques) are predominating species in obstructive airway disease research, but the new world species Callithrix jacchus (marmoset monkeys) and Saimiri sciureus (squirrel monkeys) have also been described (Hamel et al., 1986; Curths et al., 2015) (Fig. 1). Allergy-induced airway diseases have been observed in free-ranging macaques in Japan, which show rhinosinusitis after cedar pollen exposure (Hashimoto et al., 1994; Yokota et al., 1987; Sakaguchi et al., 1999). This indicates that macaques are generally susceptible to allergic reactions. Additionally, naturally Ascaris suum-sensitized macaques show positive skin prick tests and have been extensively used as inducible asthma models (Patterson and Harris, 1978; Gundel et al., 1990, 1992; Osborn et al., 1992; Turner et al., 1994; Mauser et al., 1995). To increase comparability to human allergic patients, further asthma models in nonhuman primates comprise experimental sensitization with allergens of house dust mites (HDMs, Dermatophagoides pteronyssinus and Dermatophagoides farinae; Yasue et al., 1998; Schelegle et al., 2001; Van Scott et al., 2004; Iwashita et al., 2008) and birch pollen (Bet V1 and Bet V2; Ferreira et al., 1996).

Development of novel therapeutic approaches requires proof of efficacy and toxicological evaluation of substances in appropriate animal models. Before testing in an animal species, target structures have to be analyzed for genetic homology to address efficacy in the most suitable species. Additionally, binding affinity of a test substance towards this target structure needs to be assessed. Novel test substances in asthma and COPD are often directed against immunologic targets. Among these, monoclonal antibodies are highly target specific, requiring a close homology in animal species for efficacy assessment, which is often tested in humanized mice or nonhuman primates.

Monoclonal antibodies are promising therapeutics which have been developed in nonhuman primates. Among these, IgE is an attractive target for therapeutic intervention. Human IgE-Fc has been shown to cross react with cynomolgus monkey effector cells (Saul et al., 2014), and the binding of human IgE to cynomolgus lung tissue potentially drives IgE-mediated histamine release (Ishizaka et al., 1970; Wichmann et al., 2016). Later approved anti-IgE omalizumab showed binding to cynomolgus IgE (Fox et al., 1996; Meng et al., 1996), and efficacy assessment of a human-specific anti-IgE vaccine showed a reduction of circulating IgE, indicating the general suitability to test anti-IgE therapeutics in nonhuman primates (Weeratna et al., 2016). However, preclinical safety assessment of omalizumab in nonhuman primates revealed an age-dependent reduction of platelets, which was not detected in human patients, limiting the general transferability of data obtained in nonhuman primates (EMA, 2009a; Martin and Bugelski, 2012).

Other asthma-approved monoclonal antibodies which have been preclinically analyzed in nonhuman primates include the anti-IL-5 antibodies mepolizumab (Hart et al., 2001) and reslizumab (Egan et al., 1999). Cynomolgus monkey IL-5 shows cross reactivity against humanized anti-IL-5 antibodies (Hart et al., 2001; Mauser et al., 1995). Treatment of Ascaris suum-induced airway eosinophilia was reduced by both antibodies, with sustained effects for several months (Hart et al., 2001; Egan et al., 1999). In contrast, development of the anti-IL-4 antibody pascolizumab, which had been tested for cross reactivity and safety in cynomolgus monkeys (Hart et al., 2002), was discontinued in phase II studies due to lacking effectiveness of targeting only IL-4 (Akdis, 2012).

Further monoclonal antibodies for asthma therapy targeting IL-4 receptor subunit α (dupilumab), IL-5 receptor subunit α (benralizumab), and IL-13 (lebrikizumab, tralokinumab) are currently under development in phase III studies (Tan et al., 2016; NIH, 2017; EMA, 2017). Whereas data for most of these monoclonal antibodies regarding nonhuman primate studies are not available, tralokinumab significantly inhibited BAL eosinophils and reduced AHR in a cynomolgus Ascaris model of asthma and also in a humanized mouse model (May et al., 2012). Reduction of BAL eosinophils in cynomolgus macaques was also observed in anrukinzumab (Bree et al., 2007), whereas human patients with persistent asthma did not improve upon treatment. In patients with mild atopic asthma, lung function improved by using anrukinzumab, but sputum eosinophils were not reduced (Gauvreau et al., 2011), indicating the need to carefully select patients and endpoints. In contrast to asthma, there are no approved monoclonal antibodies available for COPD therapy, for example, against IL-17; however, it is not known whether these would prevent airway neutrophilia satisfactorily (Brusselle and Bracke, 2014).

All of these approaches were investigated in old world monkeys. Nevertheless, new world monkey species also have to be considered if they show better cross reactivity than other species. For example, canakinumab only binds IL-1β (Ilaris^®, Novartis Europharm Limited, Horsham, UK) of humans and marmosets, in contrast to rodents and cynomolgus macaques, although high sequence similarity was shown. Therefore, marmosets were used for preclinical studies (Rondeau et al., 2015; EMA, 2009b). Other therapeutics applicable in obstructive lung diseases could also be investigated in new world monkeys, since, for example, roflumilast reduces neutrophilic responses in marmosets (Seehase et al., 2012).

In summary, it is important to test novel therapeutics in nonhuman primate species if targets show cross reactivity in vitro. However, transferability of results is not guaranteed, neither from rodents to NHPs (Wang et al., 2013) nor from NHPs to humans (Gauvreau et al., 2011; Martin and Bugelski, 2012). To gain insight into substance reactivity and relevant readout parameters in the target species, as much effort as possible should be made to reduce animal studies to a minimum, e.g., by applying alternative models.

The development of widely accepted alternative models for respiratory diseases, in particular in the context of inhalation of harmful substances, has lagged behind other routes of administration. This is due to the complexity of the respiratory system and diversity of local and systemic responses.

Alternative methods in respiratory research include in vitro cultures of different cell populations (monoculture, co-culture), depending on the localization within the respiratory tract, and ex vivo approaches (e.g., parenchymal strips, isolated airways, isolated perfused lung and precision-cut lung slices). In vitro cultures have been reviewed previously (Hittinger et al., 2015) and are essential tools for early steps in drug development. However, regarding NHP only limited numbers of cell lines are available, for example the rhesus macaque lung epithelial cell line 4MBr-5. The isolated perfused lung enables testing of substance deposition ex vivo and has been developed in rodents and rabbits, with rare reports in human and nonhuman primates (Byron et al., 1986; Niven and Byron, 1988; Piacentini et al., 2008; Bleyl et al., 2010; Linder et al., 1996; Briot et al., 2009; Hebb and Nimmo-Smith, 1948; de Burgh Daly et al., 1975; Nahar et al., 2013). Nevertheless, isolated perfused lungs can only be maintained for several hours, limiting extensive use.

Alternative 3-D tissue models, such as precision-cut lung slices (PCLS) are a link between in vitro and in vivo models. PCLS are fresh tissue sections of the lung. They contain all cell types that are present at the time point of preparation and reflect the functional heterogeneity within the respiratory tract. The PCLS technique has the advantage of reproducible preparation of thin tissue sections of precise thickness from one single animal or human tissue donor. Internal controls, technical replicates, references, and a variety of different concentrations of drugs and other substances can be included and tested in one donor. Moreover, PCLS offer the potential of sampling at different time points up to 2 weeks and were established in many different species, including NHPs (reviewed in Sewald and Braun, 2013; Seehase et al., 2011). They are widely used in toxicology and pharmacology. The field of application of PCLS has broadened from simply testing functional responses like airway constriction to disease modeling including calcium signaling, early allergic responses, and viral infection responses.

NHP PCLS can be analyzed regarding bronchoconstriction and mediator release. Therefore, lung sections are prepared with cross-sectioned airways. Bronchoconstriction in PCLS can be induced by different mechanisms, e.g., by histamine independent cholinergic stimulation using methacholine, mimicking lung function analyses similar to in vivo settings (Fig. 2; Seehase et al., 2011). Moreover, PCLS derived from humans and NHPs can be passively sensitized with human plasma containing IgE against a specific allergen. Incubation of sensitized PCLS with the specific allergen results in bronchoconstriction similar to EAR, potentially by a mast cell associated mechanism, since increased histamine is detected after an allergen challenge (Wichmann et al., 2016) (Fig. 3). Antihistamines and disruptive IgE inhibitors prevent this bronchoconstriction also in NHP PCLS. Marmosets show the weakest reactivity towards a disruptive IgE inhibitor, potentially due to less cross reactivity of human IgE to marmoset FcεRIα (Saul et al., 2014; Wichmann et al., 2016). Comparing data of in vivo sensitized and ex vivo sensitized marmoset PCLS reveals a reduction of initial airway area to a similar extent (Dahlmann et al., Wichmann et al., unpublished data). Interestingly, the bronchial irritant capsaicin failed to induce bronchoconstriction ex vivo in marmoset PCLS (Schleputz et al., 2012) (Wichmann et al., unpublished data).

In contrast to humans and NHPs, serotonin (5-HT) but not histamine induces bronchoconstriction in rodents (Fig. 2). In another ex vivo approach, however, 5-HT induced a constriction in electric-field-stimulated tracheal rings of in vivo challenged rhesus macaques in a 5-HT2-, 5-HT3-, and 5-HT4-receptor-dependent, and 5-HT1-receptor-independent manner (Moore et al., 2012, 2014). These findings mimic the controversial discussion about the importance of serotonin in human asthma and highlight the importance of direct comparison of human and nonhuman primate material in an experimental setting.

Mitogens such as endotoxin induces an innate immune response in fresh lung tissue ex vivo. The released mediators are highly comparable to those released in vivo. Incubation of marmoset PCLS with LPS dose-dependently increased TNF-α and MIP-1β, which were inhibited by dexamethasone and roflumilast. Likewise, TNF-α and MIP-1β were elevated in BAL of marmosets challenged with LPS in vivo, and premedication with dexamethasone and roflumilast reduced TNF-α and MIP-1β release. Additionally, TNF-α secretion correlated between human and marmoset PCLS and roflumilast treatment resulted in a highly similar maximal inhibitory concentration (Seehase et al., 2012).

In conclusion, alternative approaches offer the advantage of direct comparison to human data and reproduce data obtained in vivo. Additionally, they help to identify suitable species for further safety testing of promising drug candidates

Overall, many protocols for inductions of obstructive lung diseases in NHPs have been investigated. Macaques especially show reproducible symptoms comparable to human patients, and different biologicals have been investigated in the Ascaris asthma model. Although macaques appear to be naturally sensitive to allergens, naturally occurring allergic asthma has not been reported, indicating potential limitations of macaque models. Future therapeutic approaches point towards an allergen-specific therapy, which might need adaptation of sensitization protocols in future efficacy studies. Additionally, endpoints have to be well considered and evaluated before study onset.

To improve a successful outcome of preclinical in vivo studies, an attempt should be made to exploit the full potential of in vitro and ex vivo assays, since they are valuable tools regarding certain endpoints. Besides a direct comparison to human data, they also help to reduce animal studies. However, they cannot mimic all endpoints, e.g., the complexity of immunologic reactions. Especially for novel therapeutics targeting human-specific immunological structures, NHPs are the only appropriate species due to sequence similarity and cross reactivity. Unfortunately, efficacy and safety in preclinical NHP studies does not guarantee efficacy and safety in clinical studies, highlighting the importance of well characterized in vitro, ex vivo, and in vivo studies.

In the future, precise characterization and categorization of obstructive lung diseases in human patients is also required, and identification of early biomarkers can help to prevent chronic disease progression. Only with novel approaches can improved animal welfare and a reduction of the worldwide disease burden of obstructive lung diseases be achieved – together we can make a difference.