Advances in asthma drug discovery: evaluating the potential of nasal cell sampling and beyond.

Review

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Introduction

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Asthma and allergic rhinitis: similarities and differences through guidelines

Expert Opin. Drug Discov. Downloaded from informahealthcare.com by University of Connecticut on 04/13/15 For personal use only.

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Advances in asthma drug discovery: evaluating the potential of nasal cell sampling and beyond Luigino Calzetta, Paola Rogliani, Mario Cazzola† & Maria Gabriella Matera †

University of Rome Tor Vergata, Department of System Medicine, Rome, Italy

Nasal cells sampling and procedures for novel anti-inflammatory agents development

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Conclusion

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Expert opinion

Introduction: Inhaled corticosteroid anti-inflammatory therapy is effective at controlling disease symptoms of asthma, but a subset of patients remains symptomatic despite optimal treatment, creating a clear unmet medical need. Moreover, none of the currently available drugs for asthma are really disease-modifying or curative. Although murine models of asthma, based on transgenic and knockout animals, may offer an integrated pathophysiological system for studying the characteristics of airway inflammation and hyperresponsiveness, these alterations are noteworthily different compared with those observed in asthmatic patients. Since a clear functional and inflammatory relationship between the nasal mucosa and bronchial tissue in patients suffering from asthma and allergic rhinitis has been recognized, using preclinical models based on human nasal cells sampling might support a prompt and effective anti-inflammatory drug discovery in asthma. Areas covered: The authors provide a review, which discusses the potential role of nasal cell sampling and its application in advanced drug discovery for asthma. The contents range from the similarities and differences between asthma and allergic rhinitis up to artificial airway models based on sophisticated human lung-on-a-chip devices. Expert opinion: Nasal cell sampling and processing have reached a great potential in asthma drug discovery. The authors believe that models of asthma, which are based on human nasal cells, can provide valuable indications of proof of pharmacological and potential therapeutic efficacy in both preclinical and early clinical settings. Keywords: allergic rhinitis, artificial airways, asthma, inflammation, nasal cells Expert Opin. Drug Discov. (2014) 9(6):595-607

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Introduction

For the majority of asthmatic patients, inhaled corticosteroid anti-inflammatory therapy is effective at controlling disease symptoms, but a subset of patients remain symptomatic despite optimal treatment, creating a clear unmet medical need [1]. Moreover, none of the currently available treatments for asthma have long-term effects on airway inflammation or remodeling, and therefore, are not disease-modifying or curative [2]. Clearly, there is an urgent need for targeted, disease-modifying asthma treatments. However, the development of novel therapy for asthma has proved disappointing despite intense effort and investment. An over-reliance on animal models of allergy to define targets and expectations of efficacy, a fundamental disconnect between the directions of basic research and clinical research and the different features of asthma, bronchoconstriction, symptoms and exacerbations, which respond diversely to treatment, are three 10.1517/17460441.2014.909403 © 2014 Informa UK, Ltd. ISSN 1746-0441, e-ISSN 1746-045X All rights reserved: reproduction in whole or in part not permitted

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None of the currently available drugs for asthma are really disease-modifying or curative and a subset of asthmatic patients remains symptomatic despite optimal treatment creating a clear unmet medical need. An overestimation of murine models explains why the use of animal models of asthma by pharmaceutical companies has been disappointing in term of the lack of generation of novel treatments for asthma. A substantial functional and inflammatory relationship between the nasal mucosa and bronchial tissue in patients suffering from asthma and allergic rhinitis has been recognized. Standardized and sophisticated models of asthma based on human nasal cell challenge techniques and sensitive biomarker assays permit to effectively investigate the efficacy of novel anti-inflammatory drugs for asthma in preclinical settings. Nasal cell sampling and processing can reach the greatest potential in asthma drug discovery when adequately implemented with passive sensitization of the airway smooth muscle collected from low airway.

This box summarizes key points contained in the article.

particular issues that contribute to the challenge of identifying new therapeutics [3]. Animal models based on transgenic and knockout mice may offer an integrated pathophysiological system in which to study the characteristics of inflammation and airway alterations that are similar, but not equal, to conditions of asthmatic patients [4,5]. Unfortunately, animal models fail to reproduce all of the features of human asthma since there are noteworthy differences between human and murine models toward TH2-type inflammatory pathways, the lung structure and its development. Mice fail also to develop spontaneous symptoms, longlasting bronchoconstriction and inflammatory alterations in airway wall behavior as observed in asthmatic patients [6,7]. Access to human airway tissue has allowed development of ex vivo and in vitro models of the human airway that can be used for mechanistic studies, target identification and validation and toxicological testing [6]. These models are mainly based on cultured cells obtained from human airway tissue collected from volunteers at bronchoscopy as small biopsy samples, from lung resections as surgical waste, or postmortem [6,8]. Evidently, collecting these respiratory cells from bronchi is much more invasive and less accessible compared with harvesting respiratory cells from nasal mucosa (Figure 1). Therefore, this review will focus on the potential role of nasal cell sampling and its application for advanced drug discovery in asthma, from similarities and differences between asthma and allergic rhinitis up to artificial airway models based on sophisticated human lung-on-a-chip devices. 596

Asthma and allergic rhinitis: similarities and differences through guidelines

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Article highlights.

The Allergic Rhinitis and its Impact on Asthma (ARIA) initiative, which aims to educate and implement evidence-based management of allergic rhinitis in conjunction with asthma, and the Global Initiative for Asthma (GINA), with the purpose of reducing asthma prevalence, morbidity and mortality, represent the two worldwide initiative that allow understanding the complementarities between asthma and allergic rhinitis [9,10]. The analogies between these disorders have been suggested from the launch of these initiatives (GINA in 1993 and ARIA in 2001), and the subsequent reports and guidelines have increasingly highlighted the strict association between allergic rhinitis and asthma. To date, the most recent updated ARIA and GINA reports agree that asthma and allergic rhinitis are common comorbidities. The majority of asthmatic patients have evidence of allergic rhinitis and a significant number of patients suffering from persistent rhinitis have asthma. Both rhinitis and asthma share the same epidemiological, pathological and physiologic characteristics and are also linked by common risk factors and therapeutic approach [11,12]. A recent population-based epidemiological survey has been conducted in agreement with definitions suggested by the European Position Paper on Rhinosinusitis and Nasal Polyps [13]. This study, aimed to investigate the prevalence of asthma and its association with chronic rhinosinusitis (CRS), has demonstrated that, although geographical variation in the prevalence of asthma (5.1 -- 16.8%) was observed across Europe, asthma was more common in young adults, women and smokers and a significant association of asthma with CRS has been detected (adjusted odds ratio: 3.5) at all ages. Furthermore, the association with asthma was stronger in patients reporting both CRS and allergic rhinitis (adjusted odds ratio: 11.9) [14]. Therefore, asthma has to be suspected in patients with allergic rhinitis and asthmatic patients should be evaluated for rhinitis and, consequently, the therapeutic plans should be considered together [15]. These findings are supported by the evidence that both asthma and allergic rhinitis can be considered to be inflammatory disorders of the airways and, thus, the concept of ‘one airway, one disease’ has been suggested, and even the term ‘allergic rhinobronchitis’ has been proposed, although it has been not generally accepted [16-18]. Nevertheless, in spite of the clear association between asthma and allergic rhinitis, some distinctive characteristics may be identified such as the pathophysiological mechanisms, the clinical features and the therapeutic approach. Mainly, the nasal obstruction is essentially due to hyperaemia in rhinitis, while the enhanced bronchial contraction in asthma is predominantly mediated by the airway smooth muscle (ASM) hyperresponsiveness [19].

Expert Opin. Drug Discov. (2014) 9(6)

Accuracy of specimens for asthma models


Advances in asthma drug discovery

Local anesthesia not required

Local (nasal) anesthesia required

Local (bronchial) anesthesia required

Very easy procedures

Easy procedures

Specialist procedure Bronchial biopsy

Nasal biopsy

Nasal scraping Nasal brushing

Filter paper strips Nasal lavage Invasivity

Figure 1. Invasivity of procedures for collecting samples from respiratory airways and relative accuracy of the obtained specimens for preclinical human-based models of asthma.

However, treating allergic rhinitis decreases asthma symptoms in asthmatic patients and vice versa. Intranasal administration of corticosteroids, cromones, leukotriene (LT) modifiers, anticholinergics, the allergen-specific immunotherapy and the anti-IgE therapy are effective in both conditions via reducing the bronchial hyperresponsiveness (BHR), protecting against exacerbations of asthma and improving symptoms of rhinitis [15,20-26]. Unfortunately, there are not solid evidences on the real effectiveness of intranasal corticosteroids in improving asthmatic symptoms and, effectively, some clinical trials have shown conflicting results [27,28]. On the other hand, others drugs such as H1-antagonists and b2-agonists are selectively effective for rhinitis and asthma, respectively [20,21,29]. Compared with other treatments, allergen-specific immunotherapy represents the only treatment working on the causes of allergy and, therefore, this therapeutic approach has been recommended by ARIA guidelines for treating allergic rhinitis, although its role in adult asthma is limited [11,30]. Allergenspecific immunotherapy showed the potential of preventing asthma in children with allergic rhino-conjunctivitis, but the modest benefits must be weighed against the risk of adverse events [11]. However, there are findings suggesting that allergen-specific injection immunotherapy is highly effective in IgE-mediated diseases, via inhibiting both early and late responses to allergen exposure. Immunotherapy increases allergen-specific IgG, blocks IgE-dependent histamine release from basophils and also IgE-mediated antigen presentation to T cells. Furthermore, immunotherapy modifies peripheral and mucosal TH2 responses to allergen in favor of TH1 responses [31]. In fact, allergen-specific immunotherapy might

improve seasonal asthma and inhibit seasonal increases in BHR [32]. In any case, the evidence-based medicine moves toward managing allergic rhinitis and asthma as different manifestations of a single airway disease, rather than as two separate diseases of the nose and the lung and, therefore, combining the treatment of both these upper and lower airway conditions seems to be desirable [15,33]. In fact, the 2010 revision of ARIA clearly displayed drugs recommended for improving asthma in patients with concomitant allergic rhinitis and asthma [34]. Altogether these findings support the effort for using experimental models based on nasal cells sampling for a prompt and effective anti-inflammatory drug discovery in asthma.

Nasal cells sampling and procedures for novel anti-inflammatory agents development

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The nose represents the part of respiratory system, which is most easily accessible for morphological and pathophysiological investigations of inflammatory response to various stimuli. Therefore, considering the aforementioned correlation between inflammatory response in bronchi and nasal mucosa, collecting nasal lining fluid, mucous, cells and mucosa may represent a suitable approach for advanced modeling to be applied in novel anti-inflammatory drug discovery in asthma. Standardized and reproducible samples of both nasal exudates and mucosa cells can be obtained in a relatively noninvasive manner such as nasal lavage, filter paper strips, nasal


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brushing and scraping and nasal biopsy. All these sampling techniques may be performed before and after nasal allergen challenge (NAC) for comparing the inflammatory response, particularly in cells collected from patients suffering from allergic rhinitis. Nasal allergen challenge Recent findings have demonstrated bronchial inflammation after NAC and nasal inflammation after segmental bronchial provocation. These evidences focus on the critical link between nasal and bronchial inflammation and permit to suggest NAC a suitable model for investigating inflammatory response in asthma through nasal provocation in patients with allergic rhinitis [35-37]. NAC is performed by using solutions containing extracts of the most relevant clinical allergens. Usually, 50 -- 100 µl solution containing allergen extracts is delivered into each nostril by means of a pump spray. Acoustic rhinometry is used for detecting positive NAC reaction when the nasal volume reduction is > 30% and, subsequently, a second NAC can be performed up to 7 days after the first one [35,36]. NAC immediately induces symptoms of sneezing and itching, followed by rhinorrhea and nasal blockage. The late-phase reaction (LPR) is maximal between 6 and 24 h and resolves within 1 -- 3 days [38,39]. NAC permits to investigate novel approaches to therapy, such as the immunomodulation induced by recombinant hypoallergenic recombinant allergens. In fact, there are findings suggesting that allergen-specific immunotherapy can improve the tolerance of NAC via modifying the cellular inflammatory response assessed in nasal biopsies and the IgG levels detected in nasal lavage [40]. Furthermore, NAC allowed investigating the sublingual route of allergen-specific immunotherapy as a suitable alternative to the inoculation route for immunomodulatory therapy [41,42].


3.1

Methodologies for nasal cells sampling and processing 3.2.1 Nasal lavage 3.2

Nasal lavage consists in the introduction of 2.5 -- 5.0 ml of 0.9% NaCl physiologic solution at 37 C into the nasal cavity and its recovery after 10 s. Usually, the recovery of fluid ranges between 65 and 90% [43]. In order to decongest the nose mucosa, the intranasal instillation of an a-agonist such as oxymetazoline, a method that does not affect the quantification of mediators or biomarkers, might be necessary [44]. After that, the nasal lavage is refrigerated at 4 C and centrifugated at 400 -- 1000 g for 5 -- 20 min. The supernatant can be stored at -20 to -80 C before being analyzed and the pellet has to be resuspended in 0.5 ml PBS containing 0.1% human serum albumin for cytometry and immunostaining of cells from the nasal lining fluid [39]. Mast cells, basophils and eosinophils are normally tissue-resident cells that can be found in nasal lavage as a 598

consequence of airway recruitment due to allergic process [39]. Nasal lavage performed in subject with allergic rhinitis evidenced detectable-concentration inflammatory biomarkers such as histamine, tryptase, prostanoids, prostaglandin D2, eosinophil cationic protein, eosinophil peroxidase cysteinyl LTs, LTC4, LTD4 and LTE4, and these inflammatory biomarkers were particularly elevated after consecutive NAC [39,45-49]. On the other hand, there are conflicting evidences concerning the relevance of cytokines and chemokines, such as such as IL-3, IL-4, IL-5 and eotaxin, regulated on activation, normal T cell expressed and secreted (RANTES) and granulocytemacrophage colony-stimulating factor (GM-CSF) discerning between naturally occurred disease and NAC [50-53]. However, the concentrations of inflammatory markers are usually significantly higher in nasal lavage fluid of patients suffering from allergic rhinitis compared with normal subjects as control [54]. The validity of nasal lavage fluid analysis for assessing novel anti-inflammatory therapy in allergic rhinobronchitis has been proved by the finding that the pretreatment with topical glucocorticosteroids inhibited the inflammatory mediator release in allergic rhinitis after NAC [55]. Filter paper strips Another effective technique for recovering cytokines from inflammatory mucosa in situ is represented by the use of filter paper strips. The strips are placed on nasal turbinates for 10 min in order to adsorb nasal secretion. After that, the strips have to be washed in Hepes buffer containing 0.3% human serum albumin [56]. This method may be performed consecutively at different time after NAC in order to quantify the inflammatory response assessing inflammatory mediators. The filter paper strip method has been validated for detecting chemokines and cytokines such as IL-1b, IL-5, IL-6, IL-8, macrophage inflammatory protein-1 a, RANTES and GM-CSF at different time [56,57]. Studies on the role of cytokines during LPR have established a significant correlation between decreases in cytokine levels and symptoms after topical administration of corticosteroids in patients with allergic rhinitis, even after a single dose pretreatment [52,58]. Compared with the nasal lavage, the nasal sampling by absorption into filter paper strips gives information on both intracellular and extracellular inflammatory pathways, since the cells adhering on the paper surface may lyse and release their intracellular contents [59]. Furthermore, the filter paper strip method prevents the dilution of secretions caused by nasal lavage and, therefore, allows collecting proteins at detectable concentrations, which might be below the detection limits after nasal lavage [39]. 3.2.2

Nasal brushing and scraping Nasal cytology may be performed on samples obtained from nasal brushing and scraping. Nasal brushing can be carried out without local anesthesia. A small brush made with nylon 3.2.3



Table 1. Specific techniques for staining cytology and histology specimens from nasal cavity. Stained cells


Eosinophils Basophils Epithelial cells, nuclear and cytoplasmic changes Mast cells Neutrophils Eosinophils, neutrophils and basophils

Staining technique Hansel, Leishman and Randolph Wright and toluidine blue Papanicolaou Alcian yellow May--Grunwald Wright--Giemsa

strings is gently introduced into the nasal cavity under direct visual guidance and placed between the nasal septum and the inferior turbinate. The brush is slightly rotated while being removed and then it is washed with physiological buffer solution to tear out harvested cells [60]. Harvested cells are those floating in the nasal cavity and are mainly represented by epithelial cells. Therefore, the sample significantly reflects the cell pool involved in the barrier function of the mucous membrane [61]. Nasal scraping is performed by using a small steal or plastic curette and it allows to obtain well-preserved chunks of epithelial layer. This technique is useful for studying epithelial cells, goblet cells and granulocytes but not for cells located in deeper layer of mucosa. Also, nasal scraping does not require anesthesia and permit to obtain repeatable epithelial specimens from specific areas of nasal cavity [60]. Cells obtained with these methods can be used for morphological assays, biochemical evaluations and molecular investigations. Although nasal brushing and scraping allow evaluating the nasal inflammatory response, there are conflicting evidences on their role in adequately investigating the inflammatory processes that involve the whole thickness of the nasal mucosa. Effectively, nasal biopsy permits to study the cellular inflammatory response to challenge in all layers of the nasal mucosa [19]. Nevertheless, there are findings suggesting that well-performed nasal brushing harvests an adequate number of cells from the surface of the nasal mucosa to be used in lieu of nasal biopsies for detecting the increase of eosinophils and mast cells and for investigating the effectiveness of topical corticosteroids in patients with allergic rhinitis [62]. Furthermore, in addition to cell harvesting, nasal brushing can also provide for supernatant suitable for the quantification of pro-inflammatory cytokines [63]. Nasal biopsy Nasal biopsy is an invasive procedure that requires adequate local anesthesia achieved by applying a cotton wool plug soaked in 10% cocaine and 0.025% adrenaline below the inferior turbinate for 10 min [39,59]. It is a well-tolerated practice if it is carried out by a specialist. Although biopsy can be obtained from different locations of nasal mucosa, 3.2.4

the most common site for carrying out the biopsy is the undersurface of the inferior turbinate, just posterior to its anterior insertion to the lateral wall of the nose. This permits to standardize the sampling since significant differences in the histology of the nasal mucosa have been documented [39,64]. Biopsies including mucosal epithelium with basement membrane and submucosal tissue can be obtained under direct vision with nasal biopsy forceps [65]. The handling of small respiratory biopsies presents some specific issues such as tissue fixation, embedding and analysis that has been extensively reviewed by Jeffery and colleagues [66]. The main advantage of the nasal biopsy technique, compared with nasal brushing method, is the opportunity of studying the T and B lymphocyte response. In fact, lymphocytes tend to compartmentalize to tissue rather than migrate into the nasal lumen. Furthermore, nasal biopsy offers the opportunity to culture T cells directly from the nasal compartment and to successively investigate the inflammatory cellular response to various stimuli and the influence of novel anti-inflammatory medications on the activation and migration of lymphocytes [39,64]. While nasal brushing is suitable for studies looking at the effect of treatment on inflammatory cells within the epithelium or on epithelial cells themselves in studies involving children, large groups or multiple measures, the nasal biopsy provides a whole and comprehensive representation of the nasal mucosa and of the residential inflammatory cells. Therefore, the sampling method selected for a given study would depend on the number of requested samples and whether results obtained from epithelial layer are satisfactory [39]. 3.3

Cytology and histology evaluation Nasal cytology

3.3.1

Adequate specimens for nasal cytology may be obtained from nasal lavage, filter paper strips and nasal brushing and scraping. Cytological qualitative, quantitative and semiquantitative analysis permits to characterize the mucosal inflammatory response following specific allergenic challenge and to assay the effectiveness of treatments. In fact, the measurement of nasal cellular modifications reflects the inflammatory response of less-accessible regions of the respiratory system, such as the bronchial tree, which are involved as part of a common mucosal response in allergic and inflammatory disease [39]. As shown in Table 1, specific staining may evidence for different cells involved in inflammatory response of airways. In normal subjects, there are not significant difference in normal nasal cytology among infants, children and adults. Usually, both ciliated and goblet cells but not eosinophils and basophils are detected, although a limited number of neutrophils and bacteria may be identified [67,68]. In patients suffering from allergic rhinitis, nasal specimens contain significantly more total cells compared to healthy subjects as reported in Table 2. Although the number of basophilic cells usually correlates with nasal eosinophilia in allergic rhinitis, the nasal reactivity to NAC may also increase without a concomitant enhancement


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Table 2. Quantitative and semi-quantitative characteristics of cytology from healthy subjects and patients suffering from allergic rhinitis. Cells per HPF (103)


Normal

Semi-quantitative assay

Allergic rhinitis

Normal

Eosinophils

0

from 1 to >20

ND

Basophils Neutrophils

0 from 0 to 5

from 0.4 to >6 from 1 to >20

ND From ND to few scattered cells or small clumps

Allergic rhinitis From few scattered cells to large clumps of cells covering the entire field From few scattered cells to large number of cells From few scattered cells to large clumps of cells covering the entire field

HPF: High-power field; ND: Not detectable.

in the number of surface basophilic cells [39]. In any case, a significant increase in the number of basophilic cells of nasal lavage has been confirmed at LPR post-NAC in patients with allergic rhinitis, which has been prevented, together with the histamine release, by the pretreatment with topic administration of corticosteroids [69]. Both eosinophilic and basophilic scores are suitable for evaluating the anti-inflammatory effectiveness of novel compounds, as previously assessed by independent studies that have also ruled out the significance of neutrophils and goblet cells as cellular markers of LPR [70-72]. In addition, eosinophil and basophil influx may be used for investigating the effectiveness of long-term immunotherapy [73-75]. Nasal cytology might be also useful for validating innovative approaches to immunotherapy represented by newly designed adjuvants, such as monophosphoryl lipid A or bacteria-derived nucleotide immunostimulatory sequences, which improve the efficacy and safety of allergen immunotherapy, and for investigating alternative therapeutic strategies including the administration of allergen-derived peptides or modified recombinant allergen vaccines [31,76]. Nasal histology evaluation Immunohistochemistry of nasal biopsy allows an easy approach for amplifying the signal, identifying antibody binding to the tissue antigens and, thus, staining lymphocytes, mast cells and eosinophils. Since the image analysis is performed on two-dimensional sections, a bias in favor of higher cell counts compared with larger cells exists. However, nowadays both commercial and open-source automating cell-counter software exist that use digital imaging analysis in order to standardize the counting procedure and to normalize the image-derived data [77-80]. These softwares detect cell phenotypes in a high-throughput manner by simultaneously measuring the size, shape, intensity and texture. In situ hybridization (ISH) is a technique that enables the monitoring of gene expression in individual cells [81]. ISH is used with light microscope and it is sensitive for detecting mRNAs that accumulate in only a small population of cells within an organ [82]. ISH studies carried out for profiling cytokines of nasal biopsies evidenced an increasing of 3.3.2

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mRNA for IL-4, IL-5, IL-13 and GM-CSF, suggesting an imbalance in T-cell cytokine production in favor of a TH2-type response in patients suffering from allergic rhinitis [83,84]. Radiolabeled RNA probes are preferred for amplifying the staining of genes that are low expressed, while nonradiolabeled probes are adequately sensitive for genes that are expressed at a high level. However, non-traditional methods of probe synthesis, quantification and detection of hybridization have been proposed for appreciably improving the detection levels of non-radioactive ISH. In fact, the use of both PCR-generated templates for the synthesis of probes and the antibody sandwich technique increase the sensitivity of detection, thus reducing substrate processing time, thereby maintaining tissue morphology and providing good signal localization of non-radioactive ISH in human airway tissue [85]. Nasal cells and artificial airways Recently, several models have been proposed for the production in vitro of artificial bronchial wall by using airway tissues from bronchial biopsies, lung resections during lobectomy and postmortem [6]. Considering the significant functional and immunological relationship in terms of infiltrating leucocytes, inflammatory mediators and response to NAC between nasal and bronchial tissues, nasal sampling might be suitable for modeling artificial airways for investigating novel anti-inflammatory strategies for asthma [17,86,87]. Nasal sampling may provide for epithelial cells, fibroblasts, endothelial cells, dendritic cells, mast cells, eosinophils, basophils, macrophages, T cells and natural killer cells depending on the method used for obtaining the specimens. Residential nasal cells collected by nasal lavage, filter paper strips technique, brushing and scraping and biopsy can be grown as primary cultures and used for developing artificial airways. Monolayer cultures of both epithelial cells and fibroblasts and pseudostratified cultures of epithelial cells containing also goblet and ciliated cells can be used as monocultures [88-90]. Air--liquid interface (ALI) technique permits to closely mimic in vitro the respiratory mucosa. In fact, the availability of a huge selection of permeable supports based on 3.4




microporous membrane filters, customizable for pore size, pore density and chemical compatibility, allows modeling the three-dimensional structure of airway wall [91-93]. These models are based on co-cultures of fibroblasts and/or endothelial cells submerged at the liquid side of the system, which are interfaced with the respiratory epithelial cell layer at the air-media side through permeable supports [6,94,95]. Further complex co-cultured ALI models include also immune cells incorporated into the epithelial layer [6]. Recently several studies, carried out by ALI technique with epithelial cells isolated from patients suffering from CRS with and without nasal polyps, have permitted to investigate the relationship between the microenvironmental milieu and the host innate immunity response, the role of oxidative stress in inducing unfolding protein response and the detrimental role of interleukins in altering the sinonasal epithelial barrier [96-98]. Although these techniques are usually performed with primary respiratory cells, there are findings evidencing that experiments with ALI methods may be carried also employing immortalized cellular lines such as IMR-90 human fetal lung fibroblasts and the epithelial cell line 16HBE14o- [99,100]. Airway biopsies can be used itself as tissues explant models (TEMs) that are maintained in short-term culture for investigating the inflammatory response of respiratory mucosa [101-104], the regulatory inflammatory and immunomodulatory pathways in sinonasal mucosa of CRS patients with and without nasal polyps [105,106] and the ciliary dysfunction in subjects with CRS [107,108]. Although TEMs from nasal biopsy have the advantage of including almost all the components of the airways excluding ASM cells, the delivery of nutrients, the gas exchange and the absence of circulation within the tissue limit the length of time TEMs can be cultured for investigations of novel anti-inflammatory drugs [6]. New advances in human airway modeling, which potentially can use also cells from nasal sampling, are represented by the three-dimensional cell co-culture to organs-on-chips. This sophisticated technology requires microfabrication technologies from the microchip industry and microfluidics approaches to create cell-culture microenvironments that both support tissue differentiation and recapitulate the tissue--tissue interfaces, spatiotemporal chemical gradients and mechanical microenvironments of living organs [109]. Recently, the physiological functions and the three-dimensional microarchitecture of the whole human lung have been microengineered [109]. Human lung-on-a-chip devices constituted by airway epithelial cells and airway endothelial cells can be used for demonstrating the organ-specific features concerning airway closure and reopening, alveolar--capillary interface, surfactant production, lung inflammation and extrapulmonary absorption [110-112]. These systems may effectively mimic the human inflammatory response to cytokines and chemokines placed into the air channel and to human neutrophils that are introduced into the capillary channel. Therefore, lung-on-a-chip devices can be used to study absorption and effectiveness of novel anti-inflammatory drugs in preclinical studies [109,110].

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Conclusion

It has long been recognized that there is a substantial functional and inflammatory relationship between the nasal mucosa and bronchial tissue in patients suffering from asthma and allergic rhinitis [17,86,87]. The concept of considering asthma and allergic rhinitis as a single airway disease is in line with ARIA and GINA guidelines [11,12]. The evidence of an overestimation of murine models, even when based on transgenic and knockout mice, might explain why the use of animal models of asthma by pharmaceutical companies has been disappointing in term of the lack of generation of novel treatments for asthma [6,7,39]. This scenario offers the foundation for developing human-based models of airway that would permit to reproduce in vitro conditions of asthmatic airway. To date, several models of asthma based on cells collected from low airways are available, but a greater interest should be given also to models based on human nasal cell sampling. Effectively, nasal cells are easy to be collected and even the nasal biopsy, although being an invasive procedure, may be well tolerated when compared with small bronchial biopsy. Nasal harvesting provides for a wide range of samples such as nasal lavage, epithelial cells, connective cells, inflammatory cells and specimens of mucosa in toto. The choice of the needed specimen and, consequently, the technique for collecting cells and tissue depends on the number of samples requested for the studies and by the endpoints of the investigations to be carried out [39]. Approaching models of asthma with human nasal cells allow to investigate the effectiveness, the potency and the duration of action of novel anti-inflammatory drugs via NAC assay in subjects with allergic rhinitis, a condition that closely mimics the bronchial inflammatory response of asthmatic patients and that has been well standardized [35-39]. In addition to the classical studies based on cytological and histological evaluations of nasal cells and mucosa, to date very advanced and sophisticated models for artificial airways exist such as three-dimensional ALI and lung-on-chip models that, together with epithelium and endothelium, can incorporate also fibroblasts and inflammatory cells [91-93,109,110]. Intriguingly, the majority of cellular lines needed for these novel techniques can be obtained from nasal sampling, excluded for ASM cells. Concluding, nowadays the availability of well-standardized and sophisticated models of asthma based on human nasal cell challenge techniques, in association with the accessibility to sensitive biomarkers assays, permits to effectively investigate the efficacy of novel anti-inflammatory drugs for asthma in preclinical settings. 5.

Expert opinion

Traditionally, a drug discovery starts from the need to find new molecules able to act on a particular mechanism in a


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specific disease pathology to resolve adverse effects or to improve the effectiveness of medications. Strategies in discovering drugs have been modified over the years, from exploiting observations to rational drug design based on hormone/ neurotransmitter receptor interactions, to high-throughput screening and knowledge of the human genome. In the early days of drug discovery, pharmacology provided both target identification and validation through the modification of mechanisms and the development of animal models that respond to therapies based on these mechanisms [113]. Therefore, usually a target has to be validated before investigating candidate compounds, assuming that this will work in man if the candidate results to be safe enough to take forward into the clinic [114]. In recent years, it has become increasingly important to get as much as possible information on clinical efficacy already in the early phases of drug development. Overcoming the obstacle of preclinical experimentation, the investigated compound can enter in the clinical Phase I trial and be administered for the first time in humans to evaluate its safety and pharmacokinetic. Overall, the objectives that a clinical trial on chronic airway diseases has to reach are to achieve normal lung function, maintain normal quality of life, prevent and treat the exacerbations, prevent mortality, relieve symptoms, improve exercise tolerance, improve health status and prevent disease progression. In particular, early clinical studies of asthma should demonstrate a drug’s anti-inflammatory and/or disease-modifying properties but, unfortunately, studies in patients are logistically and economically hampered and usually require large numbers of participants during long-term observations. In contrast, several validated models for airway inflammation and responsiveness in asthma are available and represent useful tools for drug efficacy studies, either in preclinical or in clinical studies [115]. To date, several concerns have been raised against murine models of asthma and, moreover, the human-based models of asthma have been primarily developed on fluids, cells and tissues collected from low airways [4-7,39]. Unfortunately, techniques for obtaining specimens from low airways remain limited in their widespread clinical application due to their invasive nature. On the other hand, the findings assessed in this review evidence that collecting human nasal specimens such as mucous, cells and mucosa needs of procedures that are easy to be performed and relatively noninvasive. This permits to obtain an elevated number of samples that can be processed before and after NAC, especially in patients suffering from allergic rhinitis. In addition, human specimens from nasal cavity may be used for applying the most innovative OMICS technologies, such as genomic, proteomic and metabolomic investigations that might offer a great potential in the future [116,117]. In this background of technological innovation, artificial three-dimensional ALI models of airways and lungon-a-chip micro-devices expand the capabilities of nasal cell 602

culture, providing valuable low-cost alternatives to animal and clinical studies for asthma drug discovery [110]. Effectively, recently, nasal epithelial cells and ALI cultures have been employed as a tool in the preclinical development of siRNAbased therapeutics for asthma, suggesting that curettage sampling has potential in both novel investigations of disease biomarker expression and early drug development [118]. There are a few studies that compared the similarities and differences between nasal and bronchial epithelial cells, especially in the response to pro-inflammatory stimuli. Comer and colleagues suggested that nasal epithelial cells collected from patients suffering from chronic obstructive pulmonary disease (COPD) cannot substitute for in vitro bronchial epithelial cells in airway inflammation studies [119], whereas findings obtained by McDougall and colleagues in a mixed population including subjects with history of wheeze, allergic rhinitis, asthma and COPD confirmed that nasal epithelial cultures might constitute an accessible surrogate for studying lower airway inflammation [13]. In addition, although further differences exist in the extent of eosinophilic inflammation of reticular basement membrane thickness and of the epithelium shedding between bronchi and nasal in patients with allergic rhinitis, we believe that the main distinction in using models of asthma based on nasal cells instead of bronchial cells is that the former cannot provide for ASM cells [86]. In fact, ASM alterations are the more striking structural changes in asthma, which are believed to be the major determinants of BHR and the increase of ASM mass correlates with asthma severity [120,121]. Furthermore, in addition to its contractile properties, ASM may contribute to the pathogenesis of asthma by increased proliferation, and by the expression and secretion of pro-inflammatory cytokines and mediators such as RANTES, GM-CSF and monocyte chemoattractant proteins 1, 2 and 3 [122]. Therefore, adequate airway models of asthma should not exclude ASM cells. This evidence suggests the necessity of integrating the models based on nasal cells with experiments performed with bronchial tissue although, unfortunately, collecting numerous and large bronchial specimens from asthmatic patients is a matter. Nevertheless, in the past years, several in vitro and ex vivo studies have demonstrated that bronchial tissue, ASM and epithelial cells collected from subjects undergoing lobectomy and passively sensitized with serum derived from atopic asthmatic subjects with high serum IgE levels represent a suitable model of asthma [123-129]. This approach allows to reduce the problem of the availability of bronchial samples from asthmatic patients. For a satisfactory preclinical asthma drug discovery, novel compounds should be assessed also on passively sensitized precision-cut lung slices. In particular, videomicrometry of small airway slices (bronchioles < 2 mm diameter and < 1 mm thickness), processed without the complications related to the use of confounding agarose gel to inflate the lung or complex parenchymal sections that have numerous




contracting elements, might be a proper method for investigating the influence of inflammation on ASM cells hyperreactivity and, vice versa, the role of ASM cells on the inflammatory response in passively sensitized bronchioles [130,131]. Finally, nowadays, nasal cell sampling and processing have a great potential in asthma drug discovery. Especially when adequately implemented with nasal challenge models and passive sensitization of ASM cells collected from low airway, models of asthma based on human nasal cells can provide valuable indications of proof of pharmacological and potential Bibliography

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Declaration of interest The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

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Calzetta L, Passeri D, Kanabar V, et al. Brain natriuretic peptide protects against hyperreactivity of human asthmatic airway smooth muscle via an epithelial cell dependent mechanism. Am J Respir Cell Mol Biol 2014;50(3):493-501 A study that proves the relevance of models based on ASM cells and passively sensitized airway epithelial cells for investigating the bronchial hyperresponsiveness in asthma and the cross-talk between epithelium and ASM.

128.

Matera MG, Calzetta L, Parascandolo V, et al. Relaxant effect of brain natriuretic peptide in nonsensitized and passively sensitized isolated human bronchi. Pulm Pharmacol Ther 2009;22(6):478-82

129.

Matera MG, Calzetta L, Peli A, et al. Immune sensitization of equine bronchus: glutathione, IL-1beta expression and tissue responsiveness. Respir Res 2005;6:104

130.

van Lunteren E, Moyer M. Auxotonic contractile responses of rat tracheal and

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123.

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127.

Matera MG, Calzetta L, Passeri D, et al. Epithelium integrity is crucial for the relaxant activity of brain natriuretic peptide in human isolated bronchi. Br J Pharmacol 2011;163(8):1740-54


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bronchial airway smooth muscle. Pulm Pharmacol Ther 2001;14(6):443-53 A study that describes an alternative approach, compared with the classic precision-cut lung slices method, for studing small airways that is based on a videomicrometry technique that prevents the complications related to the use of confounding agarose gel to inflate the lung or complex parenchymal sections that have numerous contracting elements.

131. Wohlsen A, Uhlig S, Martin C. Immediate allergic response in small airways. Am J Respir Crit Care Med 2001;163(6):1462-9

Affiliation Luigino Calzetta1 PhD, Paola Rogliani2 MD, Mario Cazzola†2 MD, Maria Gabriella Matera3 MD † Author for correspondence 1 IRCCS, San Raffaele Pisana Hospital, Department of Pulmonary Rehabilitation, Rome, Italy 2 University of Rome Tor Vergata, Department of System Medicine, Via Montpellier 1, 00133 Rome, Italy Tel: +0039 06 2090 0631/0633; E-mail: [email protected] 3 Second University of Naples, Department of Experimental Medicine, Naples, Italy

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Drug discovery 2012 and beyond.

Potential Approaches and Recent Advances in Biomarker Discovery in Clear-Cell Renal Cell Carcinoma.

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