Evidence for vagal sensory neural involvement in influenza pathogenesis and disease.

Influenza A virus (IAV) is a common respiratory pathogen and a global cause of significant and often severe morbidity. Although inflammatory immune responses to IAV infections are well described, little is known about how neuroimmune processes contribute to IAV pathogenesis. In the present study, we employed surgical, genetic, and pharmacological approaches to manipulate pulmonary vagal sensory neuron innervation and activity in the lungs to explore potential crosstalk between pulmonary sensory neurons and immune processes. Intranasal inoculation of mice with H1N1 strains of IAV resulted in stereotypical antiviral lung inflammation and tissue pathology, changes in breathing, loss of body weight and other clinical signs of severe IAV disease. Unilateral cervical vagotomy and genetic ablation of pulmonary vagal sensory neurons had a moderate effect on the pulmonary inflammation induced by IAV infection, but significantly worsened clinical disease presentation. Inhibition of pulmonary vagal sensory neuron activity via inhalation of the charged sodium channel blocker, QX-314, resulted in a moderate decrease in lung pathology, but again this was accompanied by a paradoxical worsening of clinical signs. Notably, vagal sensory ganglia neuroinflammation was induced by IAV infection and this was significantly potentiated by QX-314 administration. This vagal ganglia hyperinflammation was characterized by alterations in IAV-induced host defense gene expression, increased neuropeptide gene and protein expression, and an increase in the number of inflammatory cells present within the ganglia. These data suggest that pulmonary vagal sensory neurons play a role in the regulation of the inflammatory process during IAV infection and suggest that vagal neuroinflammation may be an important contributor to IAV pathogenesis and clinical presentation. Targeting these pathways could offer therapeutic opportunities to treat IAV-induced morbidity and mortality.

Investigation of vagal sensory neurons in mice using optical vagal stimulation and tracheal neuroanatomy.

In rats and guinea pigs, sensory innervation of the airways is derived largely from the vagus nerve, with the extrapulmonary airways innervated by Wnt1+ jugular neurons and the intrapulmonary airways and lungs by Phox2b+ nodose neurons; however, our knowledge of airway innervation in mice is limited. We used genetically targeted expression of enhanced yellow fluorescent protein-channelrhodopsin-2 (EYFP-ChR2) in Wnt1+ or Phox2b+ tissues to characterize jugular and nodose-mediated physiological responses and airway innervation in mice. With optical stimulation, Phox2b+ vagal fibers modulated cardiorespiratory function in a frequency-dependent manner while right Wnt1+ vagal fibers induced a small increase in respiratory rate. Mouse tracheae contained sparse Phox2b-EYFP fibers but dense networks of Wnt1-EYFP fibers. Retrograde tracing from the airways showed limited tracheal innervation by the jugular sensory neurons, distinct from other species. These differences in physiology and vagal sensory distribution have important implications when using mice for studying airway neurobiology.

Regulation of vagally-evoked respiratory responses by the lateral parabrachial nucleus in the mouse.

Vagal sensory inputs to the brainstem can alter breathing through the modulation of pontomedullary respiratory circuits. In this study, we set out to investigate the localised effects of modulating lateral parabrachial nucleus (LPB) activity on vagally-evoked changes in breathing pattern. In isoflurane-anaesthetised and instrumented mice, electrical stimulation of the vagus nerve (eVNS) produced stimulation frequency-dependent changes in diaphragm electromyograph (dEMG) activity with an evoked tachypnoea and apnoea at low and high stimulation frequencies, respectively. Muscimol microinjections into the LPB significantly attenuated eVNS-evoked respiratory rate responses. Notably, muscimol injections reaching the caudal LPB, previously unrecognised for respiratory modulation, potently modulated eVNS-evoked apnoea, whilst muscimol injections reaching the intermediate LPB selectively modulated the eVNS-evoked tachypnoea. The effects of muscimol on eVNS-evoked breathing rate changes occurred without altering basal eupneic breathing. These results highlight novel roles for the LPB in regulating vagally-evoked respiratory reflexes.

Modulation of Vagal Sensory Neurons via High Mobility Group Box-1 and Receptor for Advanced Glycation End Products: Implications for Respiratory Viral Infections.

Vagal sensory neurons contribute to the symptoms and pathogenesis of inflammatory pulmonary diseases through processes that involve changes to their morphological and functional characteristics. The alarmin high mobility group box-1 (HMGB1) is an early mediator of pulmonary inflammation and can have actions on neurons in a range of inflammatory settings. We hypothesized that HMGB1 can regulate the growth and function of vagal sensory neurons and we set out to investigate this and the mechanisms involved. Culturing primary vagal sensory neurons from wildtype mice in the presence of HMGB1 significantly increased neurite outgrowth, while acute application of HMGB1 to isolated neurons under patch clamp electrophysiological investigation produced inward currents and enhanced action potential firing. Transcriptional analyses revealed the expression of the cognate HMGB1 receptors, Receptor for Advanced Glycation End products (RAGE) and Toll-like Receptor 4 (TLR4), in subsets of vagal sensory neurons. HMGB1-evoked growth and electrophysiological responses were significantly reduced in primary vagal sensory neurons harvested from RAGE deficient mice and completely absent in neurons from RAGE/TLR4 double deficient mice. Immunohistochemical analysis of vagal sensory neurons collected from mice after intranasal infection with murine pneumovirus or influenza A virus (IAV), or after intratracheal administration with the viral mimetic PolyI:C, revealed a significant increase in nuclear-to-cytoplasm translocation of HMGB1 compared to mock-inoculated mice. Neurons cultured from virus infected wildtype mice displayed a significant increase in neurite outgrowth, which was not observed for neurons from virus infected RAGE or RAGE/TLR4 deficient mice. These data suggest that HMGB1 can enhance vagal sensory neuron growth and excitability, acting primarily via sensory neuron RAGE. Activation of the HMGB1-RAGE axis in vagal sensory neurons could be an important mechanism leading to vagal hyperinnervation and hypersensitivity in chronic pulmonary disease.

The impact of influenza pulmonary infection and inflammation on vagal bronchopulmonary sensory neurons.

Influenza A virus (IAV) is rapidly detected in the airways by the immune system, with resident parenchymal cells and leukocytes orchestrating viral sensing and the induction of antiviral inflammatory responses. The airways are innervated by heterogeneous populations of vagal sensory neurons which also play an important role in pulmonary defense. How these neurons respond to IAV respiratory infection remains unclear. Here, we use a murine model to provide the first evidence that vagal sensory neurons undergo significant transcriptional changes following a respiratory IAV infection. RNA sequencing on vagal sensory ganglia showed that IAV infection induced the expression of many genes associated with an antiviral and pro-inflammatory response and this was accompanied by a significant increase in inflammatory cell recruitment into the vagal ganglia. Assessment of gene expression in single-vagal sensory neurons confirmed that IAV infection induced a neuronal inflammatory phenotype, which was most prominent in bronchopulmonary neurons, and also evident in some neurons innervating other organs. The altered transcriptome could be mimicked by intranasal treatment with cytokines and the lung homogenates of infected mice, in the absence of infectious virus. These data argue that IAV pulmonary infection and subsequent inflammation induces vagal sensory ganglia neuroinflammation and this may have important implications for IAV-induced morbidity.

Descending Modulation of Laryngeal Vagal Sensory Processing in the Brainstem Orchestrated by the Submedius Thalamic Nucleus.

The nodose and jugular vagal ganglia supply sensory innervation to the airways and lungs. Jugular vagal airway sensory neurons wire into a brainstem circuit with ascending projections into the submedius thalamic nucleus (SubM) and ventrolateral orbital cortex (VLO), regions known to regulate the endogenous analgesia system. Here we investigate whether the SubM-VLO circuit exerts descending regulation over airway vagal reflexes in male and female rats using a range of neuroanatomical tracing, reflex physiology, and chemogenetic techniques. Anterograde and retrograde neuroanatomical tracing confirmed the connectivity of the SubM and VLO. Laryngeal stimulation in anesthetized rats reduced respiration, a reflex that was potently inhibited by activation of SubM. Conversely, inhibition of SubM potentiated laryngeal reflex responses, while prior lesions of VLO abolished the effects of SubM stimulation. In conscious rats, selective chemogenetic activation of SubM neurons specifically projecting to VLO significantly inhibited respiratory responses evoked by inhalation of the nociceptor stimulant capsaicin. Jugular vagal inputs to SubM via the medullary paratrigeminal nucleus were confirmed using anterograde transsynaptic conditional herpes viral tracing. Respiratory responses evoked by microinjections of capsaicin into the paratrigeminal nucleus were significantly attenuated by SubM stimulation, whereas those evoked via the nucleus of the solitary tract were unaltered. These data suggest that jugular vagal sensory pathways input to a nociceptive thalamocortical circuit capable of regulating jugular sensory processing in the medulla. This circuit organization suggests an intersection between vagal sensory pathways and the endogenous analgesia system, potentially important for understanding vagal sensory processing in health and mechanisms of hypersensitivity in disease.SIGNIFICANCE STATEMENT Jugular vagal sensory pathways are increasingly recognized for their important role in defensive respiratory responses evoked from the airways. Jugular ganglia neurons wire into a central circuit that is notable for overlapping with somatosensory processing networks in the brain rather than the viscerosensory circuits in receipt of inputs from the nodose vagal ganglia. Here we demonstrate a novel and functionally relevant example of intersection between vagal and somatosensory processing in the brain. The findings of the study offer new insights into interactions between vagal and spinal sensory processing, including the medullary targets of the endogenous analgesia system, and offer new insights into the central processes involved in airway defense in health and disease.

Transcriptional Profiling of Individual Airway Projecting Vagal Sensory Neurons.

Bronchopulmonary sensory neurons are derived from the vagal sensory ganglia and are essential for monitoring the physical and chemical environment of the airways and lungs. Subtypes are heterogenous in their responsiveness to stimuli, phenotype, and developmental origin, but they collectively serve to regulate normal respiratory and pulmonary processes and elicit a diverse range of defensive physiological responses that protect against noxious stimuli. In this study, we aimed to investigate the transcriptional features of vagal bronchopulmonary sensory neurons using single-cell RNA sequencing (scRNA-seq) to provide a deeper insight into their molecular profiles. Retrogradely labeled vagal sensory neurons projecting to the airways and lungs were hierarchically clustered into five types reflecting their developmental lineage (neural crest versus placodal) and putative function (nociceptors versus mechanoreceptors). The purinergic receptor subunit P2rx2 is known to display restricted expression in placodal-derived nodose neurons, and we demonstrate that the gene profiles defining cells high and low in expression of P2rx2 include G protein coupled receptors and ion channels, indicative of preferential expression in nodose or jugular neurons. Our results provide valuable insight into the transcriptional characteristics of bronchopulmonary sensory neurons and provide rational targets for future physiological investigations.