Bradley J. Undem - US grants

The objective of this proposal is to develop an understanding of the functional interactions occurring between nerves and mast cells within central and peripheral airways. Experiments will be carried out to test the hypotheses that intramural nerves in isolated airways regulate the function of airway mast cells, and that mast cell associated inflammatory mediators influence neuroeffector transmission. Mast cell function in central airways will be investigated using human bronchi and guinea pig trachea and bronchi. The function of peripheral airway mast cells will be studied in isolated human airways 1-2 mm in diameter. Airway mast cell responses will be evaluated through the measurement of antigen-induced changes in smooth muscle tone and the quantification of released inflammatory mediators. Nerves in the airways will be stimulated by electric field stimulation or by stimulation of sympathetic or parasympathetic nerve trunks in the innervated guinea pig trachea preparation. Nerve function will be evaluated by measuring changes in smooth muscle tone and by examining the release of acetylcholine, norepinephrine, substance P, and vasoactive intestinal peptide. Mast cell response will be evaluated in the presence and absence of nerve stimulation and, conversely, nerve function will be evaluated in the absence and presence of antigen-induced mast cell activation. In addition, the effect of exogenous inflammatory mediators and neurotransmitters on nerve and mast cell function, respectively, will be studied. Results from the experiments will extend our knowledge of the communication existing between nerves and mast cells in the airways. Such information may provide important new insights into the pathophysiology of airway diseases such as bronchial asthma.

The objective of this proposal is to develop an understanding of the functional interactions occurring between nerves and mast cells within central and peripheral airways. Experiments will be carried out to test the hypotheses that intramural nerves in isolated airways regulate the function of airway mast cells, and that mast cell associated inflammatory mediators influence neuroeffector transmission. Mast cell function in central airways will be investigated using human bronchi and guinea pig trachea and bronchi. The function of peripheral airway mast cells will be studied in isolated human airways 1-2 mm in diameter. Airway mast cell responses will be evaluated through the measurement of antigen-induced changes in smooth muscle tone and the quantification of released inflammatory mediators. Nerves in the airways will be stimulated by electric field stimulation or by stimulation of sympathetic or parasympathetic nerve trunks in the innervated guinea pig trachea preparation. Nerve function will be evaluated by measuring changes in smooth muscle tone and by examining the release of acetylcholine, norepinephrine, substance P, and vasoactive intestinal peptide. Mast cell response will be evaluated in the presence and absence of nerve stimulation and, conversely, nerve function will be evaluated in the absence and presence of antigen-induced mast cell activation. In addition, the effect of exogenous inflammatory mediators and neurotransmitters on nerve and mast cell function, respectively, will be studied. Results from the experiments will extend our knowledge of the communication existing between nerves and mast cells in the airways. Such information may provide important new insights into the pathophysiology of airway diseases such as bronchial asthma.

C fiber; respiratory disorder; asthma; inflammation; tachykinin; selectins; neuropeptides; neurons; leukocyte adhesion molecules; gene expression; vascular endothelium; electrophysiology; leukotrienes; cytokine; disease /disorder model; vagus nerve; drug receptors; respiratory pharmacology; respiratory function; human tissue; guinea pigs;

Bronchial asthma is widely believed to be a disease involving reversible bronchospasm in inflamed airways. The disease is closely correlated with atopy and, at least in some cases, is thought to involved immunological stimulation of airway mast cells, i.e. the allergic reaction. Nerve stimulation in the airways can results in several of the signs and symptoms of asthma including bronchoconstriction, mucus secretion and inflammation. Afferent (sensory) nerves can be stimulated by a variety of environmental irritants, by cold dry air, and by exercise. Thus, rather than an immunological basis of asthma, a perversion in the neuronal control of airway function has been considered a viable hypothesis to explain the symptoms, and perhaps even cause, or certain types of this disease. This proposal is based on the concept that the neuronal and immunological theories of asthma may be integrate by the fact that mast cells and nerves functionality interact in the airways. The long term objective of this proposal is to critically define, in a mechanistic level, the functional communication between mast cells and nerves in the human airways. The major hypothesi addressed in this proposal is that antigenic stimulation of mast cells leads to a substantive and long-lasting increase in the excitability of sensory fibers in the airway wall and in the parasympathetic efferent innervation of the airways. Moreover, it is hypothesized that antigen challenge results in a phenotypical change in the sensory neuropeptide innervation of the airways. Immunohistochemical techniques together with intracellular and extracellular electrophysiological studies will be carried out to address specific hypothesis related to the mechanism of mast cell-nerve interactions. These hypotheses will be addressed in an animal model and, in some cases, human bronchial neuronal preparations. Using these techniques the anatomy and electrophysiology and function of the airway innervation will be investigated before and after immunologically stimulation of the intrinsic mast cells with specific antigen challenge. The results from this multidisciplinary approach should be of intrinsic value in providing new knowledge regarding the cellular neurophysiology of the airways. The results may also shed new light on the complex pathophysiology of asthma, and ultimately suggest new therapeutic strategies for treatment of this complex disease.

Within and just beneath the airway epithelium is a dense plexus of sensory nerve fibers. These fibers provide a protective function by initiating reflexes such as cough, vasodilation, glandular secretion, and bronchospasm. The responsiveness of the sensory fibers is not a static function. Inflammatory mediators can lead to modulation of ionic channels in the sensory nerve fibers causing changes in their excitability. In inflammatory airway disease, the sensitivity of the sensory fibers can become distorted to the extent that reflexes such as cough become irritating and non-productive. The non-productive cough is also likely to be a marker for perturbation in subconscious autonomic reflexes in peripheral airways. The inappropriate excitability of the sub-epithelial nerve plexus, may therefore lead to both the symptoms, and perhaps the pathophysiology of airway diseases. The epithelial cells are known to actively contribute to the inflammatory process by producing a variety of mediators and cytokines. Although the majority of sensory nerve fibers are within and lying just beneath the epithelium, there is surprisingly very little known about the communication between epithelial cell products and sensory nerve excitability in the airway. This proposal centers on the hypothesis that a major function of the epithelium is to recruit the nervous system in host defense mechanisms. This is accomplished by releasing mediators and cytokines that increase the excitability of the neighboring afferent nerve endings. To understand this process it is necessary to first define the phenotype of sensory nerves that innervate the epithelium. Second, it is necessary to identify ionic currents that are important in modulating the excitability of these nerve fibers. Finally, it is important to characterize the molecules in the epithelium that may serve to modulate afferent neuroexcitability. This proposal seeks to satisfy these objectives.

DESCRIPTION (provided by applicant): Activation of sensory C-fiber nerves in the airways can lead to coughing, sneezing, sensations of breathlessness, reflex mucus secretions and airway narrowing. These nerves play an important role in defending the airways from potentially damaging substances, but in inflammatory airway disease, their incessant activation may underlie many of the symptoms and much of the suffering associated the disorder. Since the classical studies by Coleridges and their colleagues in the 1970s and 80s, it has been recognized, at least at a descriptive level, that vagal C-fibers in the respiratory tract comprise at least two phenotypes, often referred to as "bronchial C-fibers" and "pulmonary C-fibers". The distinction based on the general location of the nerve terminals. We propose that it may be more useful to evaluate C-fiber phenotype based on the location of their cell bodies. One type of C-fiber is derived from neurons associated with the vagal nodose ganglion;the other C-fiber phenotype is associated more with the jugular vagal ganglia (and spinal dorsal root ganglia). Embryologically the nodose neurons are placodally derived, whereas the jugular and dorsal root ganglion neurons are derived from the neural crest. To understand the role of sensory C-fibers in health and disease, it is imperative that we develop a deeper understanding of these placodal and neural crest C-fiber phenotypes in the respiratory tract. This grant focuses attention on the idea that neural crest vs. placodal C-fiber phenotypes can be delineated not only on their activation profile, but also based on the location of their terminals within the lungs, and their neurotrophic regulation, and by the mechanisms regulating their excitability. In addition we begin to further develop the hypothesis that the reflex consequences of C-fiber activation will be strongly dependent on which C-fiber phenotype is activated. In the first aim we employ a very novel technique that we have developed that allows us to obtain detailed information regarding the relative location within the lungs of the terminals of the two C-fiber phenotypes. In the second aim we address specific hypotheses regarding relative neurotrophin regulation of the two C-fiber phenotypes in the respiratory tract. In the third aim we address the hypothesis that the excitability of the placodal C-fiber phenotype is enhanced due to express of a specific voltage-gated sodium channel (NaV1.9). In our last aim we address the hypothesis that activation of neural crest C-fiber phenotype enhances the cough reflex, whereas activation of placodal C-fibers actually inhibit the cough reflex. Each of the four aims of this grant is designed to provide novel information and insights into the neurobiology of C-fiber subtypes in the airways. We expect that this information will help build the framework from which future studies may be based that are aimed at understanding the role that these important nerves play in health and disease. PUBLIC HEALTH RELEVANCE: Activation of sensory C-fiber nerves in the airways can lead to coughing, sneezing, sensations of breathlessness, reflex mucus secretions and airway narrowing. These nerves play an important role in defending the airways from potentially damaging substances, but in inflammatory airway disease, their incessant activation may underlie many of the symptoms and much of the suffering associated with the disorder. This grant is designed to increase our understanding of the nature of sensory C-fibers in the airways so that strategies aimed at limiting the suffering associated with inflammatory airway diseases can be realized.

DESCRIPTION (provided by applicant): Respiratory virus infections modulate the sensory nervous system leading to sneezing, sore throat, coughing, reflex secretions and wheezing. For many this is a self-limiting problem; for others this can progress to significant morbidity. In fac, viral infections are the leading cause of asthma exacerbations in children, and are also a common cause of COPD exacerbation. Viral infections are also thought to be a leading cause of chronic unproductive cough that is said to affect as many as 10% of the population. The long-range goal of this proposal is to develop at a better understanding of the mechanisms and mediators involved in respiratory virus-induced sensory neuromodulation. In Aim 1 we specifically address on our hypothesis, supported by preliminary data, that viral infection leads to a phenotypic change in the vagal extrapulmonary A¿ fibers such they take on a C-fiber nociceptor-like phenotype. We focus on the nodose extrapulmonary A-fibers because they terminate just beneath the epithelium in large airways (the target cell in many respiratory virus infections) and because when they are activated it leads to coughing, reflex secretions and bronchoconstriction. We hypothesize that viral infections induce, de novo, the expression of the ligand-gated ion channels TRPV1, TRPA1, and purinergic receptors, in the A-fiber neurons rendering them responsive to myriad stimuli they would ordinarily be unresponsive to. We address this hypothesis at the level of gene expression in single identified neurons. In Aim 2 we further address this hypothesis at a functional level both electrophysiologically by recording action potential discharge from single A¿ nerve terminals in the trachea, and physiologically using the cough reflex as an outcome. In Aims 3-4 experiments are designed to address the hypothesis that the mechanisms underlying the viral-induced neuroplasticity involved brain-derived neurotrophic factor (BDNF/NT3) and/or glial cell-derived neurotrophic factor ligands (GFLs) interacting with the TRKB and GFR¿ receptors, respectively. We address our hypotheses using a strategy of mimicry, pharmacological antagonism and by making use of our recently validated method to silence gene expression in vagal sensory neurons in vivo with adeno-associated virus-sh-RNAs delivered to the nodose ganglion. The results from our multidisciplinary approach should be of intrinsic value in providing new knowledge regarding sensory neuroplasticity in the airways. The results will also shed new light on the complex pathophysiology of respiratory viral infections and possibly suggest new therapeutic strategies for treatment aimed at limiting viral evoked exacerbations of asthma, COPD, and chronic cough.

DESCRIPTION (provided by applicant): Asthma and COPD are characterized by an over-excited sensory nervous system leading to excessive reflex bronchospasm and secretions, along with persistent unproductive coughing and dyspnea that can be unmatched to lung function. These hallmark symptoms can be evoked by stimulation of nociceptors. Nociceptors are the predominate type of nerve that innervates the airways. They comprise mainly vagal afferent C-fibers and A-fibers. We have characterized three non-redundant subtypes of vagal nociceptors in the respiratory tract (a unique A¿ cough receptor and two distinct types of C-fibers). Our long-range goal is to determine the ion channels and mechanisms that underlie the excitability of these nociceptor subtypes, as well as the reflex consequences of nociceptor subtype activation. The present proposal focuses on voltage-gated sodium channels (NaVs). NaVs are critically involved in action potential generation, conduction, and in setting the threshold for nerve activation. There are 9 NaVs termed NaV1-NaV9. This proposal builds on our seminal observations that the three nociceptor subtypes in the airways express almost exclusively NaV 1.7, NaV 1.8, and NaV 1.9. These channels are not present in skeletal or cardiac muscle and are very modestly expressed in the central nervous system. This renders them ideal targets for drugs aimed at normalizing dysregulated nociception. Over the past decade, these channels have been recognized in pain nerves, leading to the rapid pharmaceutical development of selective NaV 1.7, 1.8, and 1.9 blockers. Some of these are presently in clinical trials for inflammatory and neuropathic pain. There is a dearth of knowledge about the function of these pivotal ion channels in airway nociceptors. To the extent that they have been studied in the somatosensory system, our knowledge is based largely on studies at the cell bodies in the dorsal root ganglia, as well as from behavioral studies on pain sensation. This proposal is based on our ability to study the excitability of each nociceptor subtype at the level of the nerve endings in the tissue (Aim 1) and at the level of respiratory defensive reflex consequences of their activation (Aim 2). These properties will be investigated in control animals and in animals in which we employ innovative methods to selectively eliminate, via genetic silencing or pharmacologically, NaV 1.7, 1.8, and 1.9 expression and/or function. We will do this in both mice and guinea pigs at baseline states as well as during states of hyperexcitabilty caused by inflammatory mediators (Aim 1), or in hyperreflexic states caused by respiratory virus infections (Aim 2). This work will advance our basic understanding of how the excitability of visceral nociceptor terminals are regulated in health and disease. In addition, the will provide a rational framework with which to base future clinical studies with NaV selective blockers (already in man) aimed at reducing the exacerbations and suffering of those with asthma, COPD, and chronic cough.

Abstract People with inflammatory airway disease suffer the signs and symptoms of a dysregulated vagal sensory nervous system. These signs and symptoms include inappropriate reflex bronchospasm and secretion that can severely compromise lung function, subacute, and in some cases chronic, urge to cough, and dyspnea sensations ill-matched to lung function. The vagal sensory nerves that lead to these problems are by in large the sensory nociceptive C-fibers that innervated the airways from the larynx to the alveoli. While it is clear that inflammation leads to activation of vagal C-fibers, our understanding of which inflammatory mediators are the most relevant in leading to this activation is very limited. Our understanding of the ionic mechanisms by which the inflammatory mediators activate the C-fibers is also lacking. We have developed the approach that allows us to quantify the all the genes that are expressed in the two major types of vagal C-fiber neurons that specifically innervate the respiratory tract by using deep RNA sequencing of identified neurons. In AIM 1 we will carry out such analysis and provide for the first time a complete characterization of the expression of receptors for inflammatory mediators (cytokines, chemokine, autacoids, TLRs, etc.) by airway-specific vagal C- fiber neurons. In Aim 2 we will use sate-of- the-art electrophysiological techniques to characterize the ability of activating those receptors that are expressed to evoke action potential discharge or increase the excitability of the C-fibers within the airways. In Aim 3 we will address out hypothesis that signaling evoked by stimulation of various receptors for inflammatory mediators converge on a relatively limited number of signaling molecules and ion channels to evoke activation and/or sensitization. An understanding of the profile of mediator receptors expressed by vagal C-fibers, and some of the unifying mechanisms by which these receptors lead to C-fiber stimulation will provide knowledge that will support novel therapeutic strategies aimed at limiting the suffering of those with inflammatory airways diseases such as asthma, COPD, chronic bronchitis, and pulmonary fibrosis. The new knowledge obtained will also be useful for sensory neurobiologist studying other organ systems where vagal afferent nerves contributes to the visceral pathophysiology (e.g. inflammatory bowl disease, pancreatitis).

Asthma, COPD, and chronic cough, as with other visceral inflammatory diseases, are characterized by an over-excited sensory nervous system. When airway sensory nerves are dysregulated by inflammation it can lead to excessive coughing, dyspnea, changes in breathing pattern, and reflex bronchospasm and secretions that can threaten lung function. Our long-range goal is to determine the ion channels and mechanisms that underlie the excitability of each of the sensory nerve subtypes in the airways. The present proposal focuses on voltage-gated sodium channels (NaVs). NaV are perhaps the most important ion channels regulating nerve activity as they are required for action potential generation and conduction, and are also involved in setting the threshold for nerve activation. There are nine NaV subtypes termed NaV1.1-1.9. This renewal proposal builds on our seminal observations and strong progress since the original application. We now know that the three nociceptor subtypes in the airways express almost exclusively NaV 1.7, NaV 1.8, and NaV 1.9. These channels are not present in skeletal or cardiac muscle and are very modestly expressed in the central nervous system. This renders them ideal targets for drugs aimed at normalizing an overactive airway sensory nervous system. We have completed an extensive functional analysis on the role of NaV1.7 and 1.8 in regulating each of three distinct nociceptor subtypes at the level of their terminals within the airways. In AIM I we will turn our attention to the mechanisms by which NaV1.9 regulates the excitability of these nerves. In AIM 2 we will evaluate genetically and functionally the NaV subtypes expressed in non-nociceptive RAR/SAR stretch receptive fibers. In AIM 3 we will will begin our evaluation of the role of NaV1.8 and 1.9 in the airways hyperexcitability that is associated with respiratory viral infections. In AIM 4 we will use our newly developed extrinsically innervated isolated human bronchus to evaluate, for the first time, the translatability of the major findings we obtain in laboratory animals to the human condition. We anticipate that this work will provide a conceptual and rational framework with which to base future clinical studies with selective NaV1 blocking drugs that can be applied directly to the sensory terminals in the airways with topical inhaled delivery methods.

Project Summary/ Abstract Basic and clinical research over the past decade is shedding new light on the major role played by the nervous system in respiratory pathophysiology, in particular in the ?hypertussivity? associated with chronic unproductive cough, in the ?airways hyperresponsiveness?? associated with of asthma, and in the airway narrowing and secretions associated with COPD. The evidence supports the hypothesis that these hyperactive disorders are in part secondary to dysregulation of vagal afferent C-fibers that comprise some 75% of the nerves within the respiratory tract. This grant aims to advance our understanding of the nature of the inflammatory mediators (autacoids and cytokines) responsible for activating airway C-fibers and the specific ionic mechanisms underlying this activity. This R35 will replace my two active R01 grants: R01 HL137807 ?Mechanisms of Inflammatory Activation of Vagal C-Fibers in the Respiratory Tract? and R01HL122228 ?Control of Airway Sensory Nerve Function by Voltage- Gated Sodium Channel Subtypes.? The R35 funding will allow us to go beyond the aims of the R01 grants that focus on healthy animals, and delve into the mechanisms that lead to the neuroplasticity associated with airway inflammation. In particular, we propose to evaluate the neuroplasticity in the afferent nervous system that accompanies viral infection during early life ?critical periods? with the hypothesis that such infections cause persistent neuroplastic changes. These changes lead to a hyperreactive neurophysiological state that can last into adulthood. This grant will also allow us to continue to advance more user-friendly technologies for studying airway nerves by taking advantage of modern imaging methodologies. These methods will not only serve to advance our own studies, they will also likely be exported to other laboratories interested in visceral neuroscience in general, and airway neuroscience in particular. We will also continue to advance our techniques and studies into the study of human bronchial innervation. Finally, the award will help keep the path paved for continued mentoring of young investigators interested in pursuing airway neuroscience research.