Neuropeptides in asthma, chronic obstructive pulmonary disease and cystic fibrosis

In asthmatic airways, mucus content positively correlated with SubP expression; NK1 receptors were also elevated with strongest expression detected on goblet cells [81]. The authors of that study concluded that neurogenic mechanisms contributed to asthma. Consistent with that, Tomaki and colleagues found that SubP levels in sputum correlated with airway obstruction in asthma [82]. In an experimental murine model of allergic asthma, increased bronchoalveolar lavage fluid concentrations of SubP were associated with induction of muc5AC mRNA [83], further suggesting a potential pathogenic role for tachykinins in asthma. Other studies have found a positive correlation between muc5AC and NKA protein expression in the sputum of asthmatics [84]. There are currently no studies available that provide information regarding the role of NKB in regulating muc5AC or muc5B expression, nor of SubP or NKA in the regulation of muc5B, in asthma. However, given that muc5AC is increased and muc5B decreased in the sputum obtained from asthmatics [48], it is possible (although speculative) that tachykinins have stimulatory roles on muc5AC, but inhibitory roles on muc5B.

Therapeutically, tachykinin receptor antagonists have been explored in asthma, with some reports suggesting beneficial effects (reviewed here [85]). For such studies, bronchoconstriction, inflammation and lung function were primary endpoints. For example, Van Schoor and colleagues found that SR 48969, a NK2 receptor antagonist, prevented bronchoconstriction provoked by NKA in mild asthmatics [86]. However, more recently, the NK1/NK2 receptor antagonist AVE5883 has provided mixed results, in which it mitigated NKA-mediated bronchoconstriction, but augmented allergen-induced decreases in forced expiratory volume (FEV₁) in people with asthma [87]. Because decreases in FEV₁ could be due to either bronchoconstriction and/or mucus obstruction, it is possible that dual blockade of NK1 and NK2 receptors in asthmatics augmented mucus secretion and/or bronchoconstriction to allergen. Therefore, examining expression of muc5AC and muc5B in the sputum of people with asthma provided tachykinin receptor antagonists might be especially important. Finally, despite some evidence that tachykinin receptor antagonists might be beneficial, they have failed to reach the market for asthma [88].

One report suggests that the concentration of SubP is elevated in the sputum of COPD patients [89], and it was suggested that neurogenic inflammation might contribute to the airway narrowing in COPD. Interestingly, however, in individuals with COPD, sputum SubP and NKA decreased during exacerbation [90]. The authors proposed that continual stimulation of sensory nerves during an exacerbation might have led to neuropeptide depletion, thus accounting for the decreased content. Additional studies have shown that cigarette smoke induces mucus secretion of goblet cells through activation of sensory nerve fibers [91], with SubP being the proposed mediator. Thus, since smoking is a major risk factor for COPD [92, 93], these findings suggested that tachykinin antagonists might be beneficial for COPD [29]. Furthermore, if SubP does mediate cigarette-induced secretion of goblet cells, then it is possible that it might also contribute to increased expulsion of mucus in COPD airways and/or regulate muc5AC and muc5B expression.

Therapeutically, tachykinin receptor antagonists have been shown to decrease inflammation in animal models of COPD [94]. Specifically, the number of macrophages and dendritic cells was decreased in the lung lavage fluid of mice exposed to cigarette smoke and provided the tachykinin receptor antagonist AVE5883. It is also interesting to note that several studies have found a pro-relaxing effect of SubP and NKA human blood vessels [95]. From this perspective, tachykinin antagonists might be of detriment in subpopulations of people with COPD (e.g., those that have pulmonary hypertension). This might explain why none of the tachykinin receptor antagonists that were in development for COPD [96] have exhibited a clear therapeutic benefit.

Although SubP stimulates gland secretion in “normal” submucosal glands, reports suggest that it is ineffective in people with CF [76]. This finding has been reproduced in pigs with CF [97]. An implication from those studies was that defective responses to SubP might contribute to airway pathology in CF. Given that glandular secretion in response to SubP is defective in CF, a speculation is that SubP-mediated secretion in CF might be associated with enhanced muc5AC to muc5B secretion ratios, effectively mimicking asthma [98]. Moreover, in contrast to asthma and COPD, where enhanced SubP and/or tachykinin signaling is potentially pathogenic, a decrease in SubP-mediated secretion in CF might be of detriment [99]. However, restoring SubP-mediated signaling through tachykinin agonists seems like an ineffective strategy in CF because evidence suggests that the defect in SubP-mediated glandular secretion in CF is due to loss of CFTR [100]; thus, CFTR correctors and/or modulators would be required. Even if such a strategy was pursued, the pro-inflammatory effects [101] and potential bronchoconstricting effects [72‐75] of tachykinins make this a less appealing option.

Some evidence suggests a functional loss of VIP-innervation to the airways in asthma [116], although this may be secondary to inflammation. It was speculated that the loss of VIP-innervation to the airway diminished the amount of bronchodilation mediated by the nervous system. Athari et al. also found that mucus hypersecretion and muc5AC mRNA were significantly decreased when enzyme-degradation resistant VIP was delivered to the airways of mice with experimentally induced asthma [117]. Although speculative, this finding might suggest that VIP has an inhibitory role on muc5AC expression in asthma. However, perhaps inconsistent with this speculation is data from the pancreas suggesting that VIP increases muc5AC expression [118]. Although we were not able to find any studies concerning the effects of VIP on muc5B regulation and/or expression in asthma, since muc5B might be decreased in asthma [119], and VIP might also be decreased, then one speculation is that VIP directly stimulates production of muc5B.

In 2003, Linden and colleagues examined the effects of a VIP agonist in asthma and found a short, but effective, bronchodilatory effect [120]. Others have shown that VIP possesses potent anti-inflammatory effects and inhibits eosinophil migration [121]. These properties make it an appealing therapeutic candidate for asthma. However, due to short plasma half-lives, VIP analogs have been met with limited enthusiasm clinically [122]. Efforts to modify formulation and delivery have been ongoing. Very recently, one agonist with sustained release (PB1046) has undergone further development for pulmonary hypertension [123]. Perhaps a renewed interest in VIP analogs will reinvigorate efforts to examine their therapeutic potential in asthma.

Increased serum levels of VIP might be a marker of acute exacerbations in COPD [124], although whether they are a cause or consequence is unknown. An argument for consequence is derived from studies suggesting a beneficial effect of inhaled VIP on quality of life in COPD [125], as well as evidence indicating a protective role of VIP against pulmonary hypertension in COPD. However, an argument for causal relationship might be inferred from studies demonstrating that chronic bronchitis, which is a common feature of COPD [126], is associated with increased VIP innervation to the mucus glands [127]. The authors suggested that increased VIP innervation to the mucus glands was linked to increased sputum production.

No studies were available that described VIP-mediated activation of goblet cells or glands in either humans or animal models of COPD. Similarly, we were unable to identify any studies that examined the effects of VIP on muc5AC or muc5B expression in COPD airways. However, receptors for VIP (vasoactive intestinal peptide receptor 1 (VPAC), vasoactive intestinal peptide receptor 2 (VPAC2)) were elevated in the epithelium and glands in biopsies from smokers with chronic bronchitis [128]. This finding might suggest that regulation of mucus secretion by VIP is altered in COPD. Additionally, since CFTR dysfunction is involved in COPD pathogenesis [56], one speculation is that VIP-mediated submucosal gland secretion, which is dependent upon CFTR [129], is impaired in COPD.

Very few studies have examined the therapeutic potential of VIP in COPD. As highlighted above, PB1046 is a VIP agonist with sustained release that is being developed for pulmonary hypertension [123]. Because pulmonary hypertension is common in COPD [130], it is possible that PB1046 will be of clinical value in COPD. However, given that increased VIP innervation to the mucus glands in COPD is proposed to contribute to increased sputum production [126], VIP agonists might be of mixed benefit.

Studies suggest that VIP nerve distribution and density are decreased in the airway epithelium, submucosal glands, alveolar walls and blood vessels of people with CF [131]. The decrease in VIP distribution was proposed to be secondary to infection and inflammation. It has also been shown that CF airway glands do not respond to VIP stimulation [129]. This lack of response has been interpreted to indicate that VIP stimulates gland secretion through CFTR-dependent mechanisms [132]. The authors suggested that the lack of VIP-mediated gland secretion might promote a hyper-inflammatory airway environment that contributes to CF lung disease. Interestingly, in mice, lack of VIP evokes CFTR dysfunction, creating a CF-like disease [133]. Together, these finding suggests a potential reciprocal relationship between VIP signaling and CFTR function.

Since VIP is ineffective at stimulating gland secretion in CF, then it seems an unlikely candidate to mediate the increased muc5B concentrations (which is largely expressed in the glands and to a lesser extent in surface goblet cells [134]) that are observed during CF exacerbations [135]. It is also unlikely that VIP agonists would be therapeutically beneficial for restoring glandular secretion in CF, since VIP-mediated secretion is dependent upon CFTR [132]. Thus, co-administration with CF correctors or potentiators would be necessary. However, the proposed anti-inflammatory and bronchodilator properties of VIP [121] make it a therapeutic option worth exploring.

CGRP has long been suspected for having important modulatory role in asthma, due to its airway constricting capacity [146, 147]. Indeed, reports suggest that CGRP is increased in the bronchoalveolar lavage fluid of asthmatics and might contribute to the late phase asthmatic reactions following provocation by allergen inhalation [148]. CGRP-positive nerve fibers are also increased in animal models given viral infections, which are risk factors for asthma [149]. Interestingly, Larson and colleagues demonstrated that CGRP is expressed ectopically in mucus cells of ovalbumin (OVA)-sensitized Brown-Norway rats [150]. They suggested that this accumulation of CGRP might represent an additional releasing mechanism involved in quick hypersensitivity responses and mucus secretion. A recent investigation by Sui and colleagues found that a combination of CGRP and gamma-aminobutyric acid (GABA) are responsible for goblet cell hyperplasia and muc5AC induction in a murine model of asthma [39]. Using the same model, they also found that elimination of pulmonary neuroendocrine cells, which express CGRP, decreased the expression of goblet cells and muc5B.

There are currently several small molecules, as well as a monoclonal antibodies [151], that inhibit CGRP signaling [152]. Studies exploring the therapeutic potential of inhibiting CGRP signaling have focused largely on the cardiovascular system [153‐156], with an emphasis on migraines. Many of those studies demonstrated an acute beneficial effect, however, liver toxicity associate with frequent use of CGRP small molecule inhibitors has hampered progress. To the best of our knowledge, there are no clinical studies that have examined the potential of CGRP receptor antagonists in clinical populations of asthmatics. However, with several tools available, examining inhibition of CGRP in the context of asthma should be achievable, even if only on a small scale. Based upon studies in animals, it is expected that inhibition of CGRP would alleviate some of the mucus phenotypes in allergic asthma [39].

Increased concentrations of CGRP have been identified in the sputum of people with COPD [157], where they have been speculated to play a role in promoting airway inflammation. Similarly, Gu et al. found increased frequency of CGRP-positive cells in the airways of people with COPD but decreased epithelial expression of CGRP receptors [158]. Although there are no studies that have specifically examined the effects of CGRP on muc5AC or muc5B expression in COPD, finding altered CGRP receptor expression in COPD airway epithelial cells suggests a possible influence of CGRP on muc5AC and muc5B expression. Correlative data also demonstrates a relationship between COPD, notch signaling, muc5AC mRNA, and pulmonary neuroendocrine cells (which synthesize CGRP among many other neuropeptides) [159].

We carefully examined the literature but were unable to find any studies that assessed the therapeutic potential of CGRP antagonists/blockers in preclinical or clinical COPD studies. However, given that CGRP can stimulate smooth muscle contraction in human airways [160], and has been implicated in mucus secretion in normal airways [142, 143], it is predicted that inhibition of CGRP might be of benefit in COPD. Yet, studies indicating that CGRP agonists possess specific anti-inflammatory properties, as well as vasodilatory properties [138], might suggest that inhibiting CGRP could be of potential negative consequence in COPD. Thus, clinical and/or preclinical studies that address the potential benefits of blocking CGRP (or the potential negative consequences) in COPD are necessary.

Although CGRP has also been shown to increase submucosal gland secretions in non-CF airways, it is an ineffective secretagogue in CF airways [161]. Of note, increased CGRP content has been found in the airway submucosal glands of humans with CF, as well as in mice, ferrets, and pigs with CF [161]. Taken together, these findings suggest that elevated CGRP in CF airways might be a compensatory mechanism to counter the lack of CGRP-mediated submucosal gland secretion. The authors also demonstrated an important role for CGRP in maintaining airway progenitor cells, which they speculated might be important for lung injury and repair in CF airways. Since CGRP participates in goblet cell hyperplasia and muc5AC induction in other disease models [39], it is possible that it is also involved in CF exacerbations, in which increased muc5AC has been noted [135].

With that said, it is difficult to predict whether inhibiting CGRP would be of significant benefit in CF, as one might anticipate mixed results (e.g. anti-inflammatory and vasodilatory properties versus involvement in mucus secretion). However, in the specific context of mucus, inhibition of CGRP might be of benefit in CF. Given the numerous CF animal models [162‐165], preclinical studies examining this possibility are feasible.

The role of NPY in asthma remains unclear. For example, NPY has been associated with stress-induced exacerbation in asthma [175‐177]. From those studies, it was suggested that NPY facilitated inflammation, as its concentrations correlated with the number of leukocytes and eosinophils in the bronchoalveolar lavage fluid. Similarly, NPY through activation of its Y1 receptor, was demonstrated to be critical for the development of allergic airway inflammation in mice [178]. Specifically, NPY and Y1 receptor knock-out mice showed decreased eosinophils in the bronchoalveolar lavage fluid, as well as decreased circulating immunoglobulin E (IgE) levels, compared to wild-type mice under allergic conditions [178]. Additional studies have found that loss of forkhead box protein P1 (Foxp)1 and forkhead box protein P4 (Foxp4) in mice can induce ectopic expression of NPY in airway epithelia, resulting in airway hyperresponsiveness [172]. Loss of Foxp1 and Foxp4 are also associated with ectopic expression of muc5AC in the airway [179]. Thus, although a direct regulation of muc5AC by NPY has not been established, the two parallel ectopic expressions, suggest an association. Consistent with that, elimination of pulmonary neuroendocrine cells in a murine model of asthma, decreased Npy gene expression, which was associated with decreased goblet cell hyperplasia [39]. However, in contrast to the aforementioned studies, Chanez and colleagues found decreased expression of NPY in the airway smooth muscle, but not in the epithelium, of people with asthma [180]. The authors proposed that NPY might be protective in the airway, and that loss of NPY might contribute to mucus hypersecretion. Similarly, Lacroix and Mosimann reported that pretreatment with NPY decreased nasal obstruction and mucus secretion in allergic rhinitis [181].

Although the NPY modulation has been explored in obesity, alcoholism, anxiety, depression, epilepsy and pain (reviewed in [34, 182, 183]), its potential benefit in asthma is largely unknown and unexplored. The numerous small molecules that have been developed to either potentiate or inhibit NPY receptor offer promise for investigating NPY signaling in asthma [182].

In COPD, it has been reported that NPY expression is decreased in the lung epithelium, glands and smooth muscle tissue [171], and the authors speculated that such changes might contribute to COPD pathogenesis. In mice, secondary tobacco smoke exposure, which is considered a risk factor for COPD [184], increases the density of the NPY nerve fibers in the tracheal smooth muscle [185]. The increase in NPY nerve fibers was associated with airway hyperresponsiveness, suggesting a potential pathological role for NPY. Others have shown that NPY stimulates secretion of interleukin 6 from airway fibroblasts [186]. An implication therefore was that NPY might contribute to diseases like COPD and asthma, in which elevated interleukin 6 has been found in the sputum [187]. Interestingly, reports suggest that the concentration of muc5AC is increased in the sputum of smokers [188]; thus, one speculation is that NPY expression is positively correlated with muc5AC. Unfortunately, no studies have directly examined the effects of NPY of submucosal gland secretion or goblet cell degranulation, in COPD.

To address the role of NPY in COPD, studies utilizing antagonists or agonists in animal models or humans with COPD are required. Additionally, although no studies have examined the effects of modulating NPY in COPD, the NPY antagonist BIBO 3304 has purported beneficial effects in pulmonary hypertension [189]. Therefore, it is possible that NPY modulation might be of benefit in some people with COPD, such as those that have pulmonary hypertension [130].

NPY was of interest in CF due to its close chromosomal location to CFTR [190]. However, a linkage between the loci of CFTR and NPY was excluded, and thus it was concluded that NPY likely played little role in CF pathogenesis. The only other study that has examined NPY in CF was conducted in the olfactory system, in which it was found that NPY was increased in the olfactory epithelium of CF mice [191]. The increased NPY was associated altered expression of specific microvilli proteins, and it was suggested that olfactory function might be affected. Thus, whether NPY is involved in CF pathogenesis, or modulates mucus secretion in CF, remains unknown. The numerous CF animal models that develop airway disease similar to humans [162‐165], as well as the multiple small molecules available that inhibit or potentiate NPY signaling [182, 189] offer ample opportunities to investigate the potential role of NPY in CF.

Recently, work by Sun and colleagues showed that the number of bombesin positive cells was increased in the airways of asthmatics [39]. Similarly, there have been several studies suggesting that exposure to tobacco products, which are triggers for asthma [202], increase components of BLP signaling in the lungs [203, 204]. Interestingly, the increased expression of BLP signaling components persisted after smoking ceased, suggesting a long-term sensitivity to the proliferative effects of BLPs. Additionally, bronchopulmonary dysplasia, in which bombesins have been implicated [205], is considered a risk factor for asthma [206]. Evidence also suggests that bombesin receptor-activated protein regulates muc5AC hypersecretion through neutrophil elastase [207]; neutrophil elastase been implicated in goblet cell degranulation and mucus hypersecretion in a rodent model of asthma [208]. While we found no studies describing the effects of bombesins on submucosal gland activation and/or regulation of muc5B in asthma, its ability to modulate glycoprotein secretion from whole tracheas under normal physiological conditions [201] suggests a potential role for it in pathophysiological conditions. Finally, bombesin antagonists have not been explored in asthma; however, several small peptide antagonists have been developed [209]. For example, monoclonal antibodies targeted against GRP have shown promise for disrupting bombesin signaling in human airways. Thus, using these tools to disrupt bombesin signaling might shed new light onto the role that they play in asthma.

Bombesins have been implicated in COPD [193], with evidence suggesting that there is greater release of bombesins from COPD airways. Consistent with that, greater numbers of pulmonary neuroendocrine cells, which express bombesins, have been observed in the airways of people with COPD [158]. Potential consequences of increased bombesin signaling in COPD include inflammation [210], bronchoconstriction [211], and altered lung injury repair [212]. Additionally, bronchopulmonary dysplasia, in which bombesins have been implicated, may increase the risk for development of COPD later in life [213]. Although the role of bombesins in modulating mucus secretion or muc5AC/muc5B regulation in COPD is unknown, given that bombesins are increased in COPD [193] and muc5B is also increased [188], then it is possible that either a direct or indirect relationship between bombesin signaling and muc5B expression exists. Bombesin antagonists have not been explored in COPD, and therefore preclinical studies focused on inhibiting bombesins might be of particular interest.

Increased expression of bombesin in neuroendocrine cells of people with CF has been reported [214, 215]. While no mechanisms explaining those changes were suggested, additional studies have shown that neuroendocrine cells express CFTR [43]. An implication therefore is that CFTR function is directly linked to pulmonary neuroendocrine function. Thus, given the proposed importance of neuroendocrine cells in asthma and COPD, it is possible that an undiscovered yet important role of neuroendocrine cells, and by extension bombesins, in CF exists. This might be especially true given their proposed involvement in submucosal gland secretion [201], muc5AC expression [207], inflammation [210], bronchoconstriction [211], and lung injury repair [212]. Utilizing available bombesin antagonists to explore the role of bombesins in CF might open the door to new therapeutics.

Very few studies have examined granins in asthma. One study reported increased serum levels of chromogranin A in a mixed population of individuals, some of which had asthma [218]. The premise of that study was to identify biomarkers of pulmonary neuroendocrine tumors. Recently, Sui and colleagues reported increased chromogranin A in the airways of mice sensitized to ovalbumin [39]. Elshafie and colleagues also reported that increased wheezing in a patient with asthma was associated with increased serum levels of chromogranin A [219]. It has also been reported that secretogranin II (known as chromogranin C), is a potent chemoattractant for eosinophils [220], which play a critical role in asthma [221]. The proposed signaling molecule mediating eosinophil chemotaxis in response to secretogranin II was cyclic AMP [220].

While we were not able to find any studies examining the role of granins in mucus hypersecretion in asthma, secretoneurin induces mucus hypersecretion in non-asthmatic human airway cells [36]. Moreover, studies suggesting that secretoneurin is co-released with SubP and CGRP raise the possibility that secretoneurin is pro-inflammatory [222]. If true, then inhibiting secretoneurin might be particularly useful in asthma. Since the cell surface receptor secretoneurin and other granins are currently unknown (Fig. 2), future studies focused on identifying the receptor responsible for granin signaling are important [223].

Elevations in serum levels of chromogranin A have been reported in people with COPD, and interestingly, the degree of obstruction correlated with chromogranin A concentrations [224]. The authors concluded that elevated chromogranin A reflected ongoing inflammatory processes in the airway. Circulating secretoneurin concentrations were also measured in people with COPD; compared to those with acute heart failure, people with COPD had decreased levels of secretoneurin [225]. It was proposed that secretoneurin was a “protective mediator” in people with heart failure. To the best of our knowledge, no studies have examined the potential contribution of granins in mucus-related phenotypes in COPD. However, it is possible that granins contribute to airway obstruction in COPD [224] through modulation of inflammation [222] and/or mucus secretion [36]. Additional studies are required to explore these possibilities.

Data indicate that pulmonary neuroendocrine cells expressing chromogranin A also express CFTR [226]. In neuroendocrine cells with diminished CFTR, neurosecretory properties were altered [226]. In follow-up study, Pan and colleagues determined that the number of pulmonary neuroendocrine cells were decreased in a mouse model of CF, resulting in diminished oxygen sensing [67]. An implication from those studies was that loss of CFTR might impair the function of neuroendocrine cells that express chromogranin A, and possibly alter chromogranin A expression and/or signaling. While we were unable to find any additional studies that investigated granins in CF, their potential for modulating inflammation [222] and mucus secretion [36] suggest that these neuropeptides might be of interest in CF airway disease.