Peroxidase-mediated mucin cross-linking drives pathologic mucus gel formation in IL-13-stimulated airway epithelial cells.

Mucus plugs occlude airways to obstruct airflow in asthma. Studies in patients and in mouse models show that mucus plugs occur in the context of type 2 inflammation, and studies in human airway epithelial cells (HAECs) show that interleukin 13 (IL-13) activated cells generate pathologic mucus independently of immune cells. To determine how HAECs autonomously generate pathologic mucus, we used a magnetic microwire rheometer to characterize the viscoelastic properties of mucus secreted under varying conditions. We found that normal HAEC mucus exhibits viscoelastic liquid behavior and that mucus secreted by IL-13 activated HAECs exhibits solid-like behavior caused by mucin cross-linking. In addition, IL-13 activated HAECs show increased peroxidase activity in apical secretions, and an overlaid thiolated polymer (thiomer) solution shows an increase in solid behavior that is prevented by peroxidase inhibition. Furthermore, gene expression for thyroid peroxidase (TPO), but not lactoperoxidase (LPO), is increased in IL-13 activated HAECs and both TPO and LPO catalyze the formation of oxidant acids that cross-link thiomer solutions. Finally, gene expression for TPO in airway epithelial brushings is increased in asthma patients with high airway mucus plug scores. Together, our results show that IL-13 activated HAECs autonomously generate pathologic mucus via peroxidase-mediated cross-linking of mucin polymers.

A novel DNase assay reveals low DNase activity in severe asthma.

Secreted deoxyribonucleases (DNases), such as DNase-I and DNase-IL3, degrade extracellular DNA, and endogenous DNases have roles in resolving airway inflammation and guarding against autoimmune responses to nucleotides. Subsets of patients with asthma have high airway DNA levels, but information about DNase activity in health and in asthma is lacking. To characterize DNase activity in health and in asthma, we developed a novel kinetic assay using a Taqman probe sequence that is quickly cleaved by DNase-I to produce a large product signal. We used this kinetic assay to measure DNase activity in sputum from participants in the Severe Asthma Research Program (SARP)-3 (n = 439) and from healthy controls (n = 89). We found that DNase activity was lower than normal in asthma [78.7 relative fluorescence units (RFU)/min vs. 120.4 RFU/min, P < 0.0001]. Compared to patients with asthma with sputum DNase activity in the upper tertile activity levels, those in the lower tertile of sputum DNase activity were characterized clinically by more severe disease and pathologically by airway eosinophilia and airway mucus plugging. Carbamylation of DNase-I, a post-translational modification that can be mediated by eosinophil peroxidase, inactivated DNase-I. In summary, a Taqman probe-based DNase activity assay uncovers low DNase activity in the asthma airway that is associated with more severe disease and airway mucus plugging and may be caused, at least in part, by eosinophil-mediated carbamylation.NEW & NOTEWORTHY We developed a new DNase assay and used it to show that DNase activity is impaired in asthma airways.

Utility of eosinophil peroxidase as a biomarker of eosinophilic inflammation in asthma.

BACKGROUND: The relative utility of eosinophil peroxidase (EPX) and blood and sputum eosinophil counts as disease biomarkers in asthma is uncertain. OBJECTIVE: We sought to determine the utility of EPX as a biomarker of systemic and airway eosinophilic inflammation in asthma. METHODS: EPX protein was measured by immunoassay in serum and sputum in 110 healthy controls to establish a normal reference range and in repeated samples of serum and sputum collected during 3 years of observation in 480 participants in the Severe Asthma Research Program 3. RESULTS: Over 3 years, EPX levels in patients with asthma were higher than normal in 27% to 31% of serum samples and 36% to 53% of sputum samples. Eosinophils and EPX correlated better in blood than in sputum (rs values of 0.74 and 0.43, respectively), and high sputum EPX levels occurred in 27% of participants with blood eosinophil counts less than 150 cells/µL and 42% of participants with blood eosinophil counts between 150 and 299 cells/µL. Patients with persistently high sputum EPX values for 3 years were characterized by severe airflow obstruction, frequent exacerbations, and high mucus plug scores. In 59 patients with asthma who started mepolizumab during observation, serum EPX levels normalized in 96% but sputum EPX normalized in only 49%. Lung function remained abnormal even when sputum EPX normalized. CONCLUSIONS: Serum EPX is a valid protein biomarker of systemic eosinophilic inflammation in asthma, and sputum EPX levels are a more sensitive biomarker of airway eosinophilic inflammation than sputum eosinophil counts. Eosinophil measures in blood frequently miss airway eosinophilic inflammation, and mepolizumab frequently fails to normalize airway eosinophilic inflammation even though it invariably normalizes systemic eosinophilic inflammation.

Exploring antiviral and anti-inflammatory effects of thiol drugs in COVID-19.

The redox status of the cysteine-rich SARS-CoV-2 spike glycoprotein (SARS-2-S) is important for the binding of SARS-2-S to angiotensin-converting enzyme 2 (ACE2), suggesting that drugs with a functional thiol group ("thiol drugs") may cleave cystines to disrupt SARS-CoV-2 cell entry. In addition, neutrophil-induced oxidative stress is a mechanism of COVID-19 lung injury, and the antioxidant and anti-inflammatory properties of thiol drugs, especially cysteamine, may limit this injury. To first explore the antiviral effects of thiol drugs in COVID-19, we used an ACE-2 binding assay and cell entry assays utilizing reporter pseudoviruses and authentic SARS-CoV-2 viruses. We found that multiple thiol drugs inhibit SARS-2-S binding to ACE2 and virus infection. The most potent drugs were effective in the low millimolar range, and IC50 values followed the order of their cystine cleavage rates and lower thiol pKa values. To determine if thiol drugs have antiviral effects in vivo and to explore any anti-inflammatory effects of thiol drugs in COVID-19, we tested the effects of cysteamine delivered intraperitoneally to hamsters infected with SARS-CoV-2. Cysteamine did not decrease lung viral infection, but it significantly decreased lung neutrophilic inflammation and alveolar hemorrhage. We speculate that the concentration of cysteamine achieved in the lungs with intraperitoneal delivery was insufficient for antiviral effects but sufficient for anti-inflammatory effects. We conclude that thiol drugs decrease SARS-CoV-2 lung inflammation and injury, and we provide rationale for future studies to test if direct (aerosol) delivery of thiol drugs to the airways might also result in antiviral effects.

Thiol drugs decrease SARS-CoV-2 lung injury in vivo and disrupt SARS-CoV-2 spike complex binding to ACE2 in vitro.

Neutrophil-induced oxidative stress is a mechanism of lung injury in COVID-19, and drugs with a functional thiol group ("thiol drugs"), especially cysteamine, have anti-oxidant and anti-inflammatory properties that could limit this injury. Thiol drugs may also alter the redox status of the cysteine-rich SARS-CoV-2 spike glycoprotein (SARS-2-S) and thereby disrupt ACE2 binding. Using ACE2 binding assay, reporter virus pseudotyped with SARS-CoV-2 spikes (ancestral and variants) and authentic SARS-CoV-2 (Wuhan-1), we find that multiple thiol drugs inhibit SARS-2-S binding to ACE2 and virus entry into cells. Pseudoviruses carrying variant spikes were less efficiently inhibited as compared to pseudotypes bearing an ancestral spike, but the most potent drugs still inhibited the Delta variant in the low millimolar range. IC50 values followed the order of their cystine cleavage rates and lower thiol pKa values. In hamsters infected with SARS-CoV-2, intraperitoneal (IP) cysteamine decreased neutrophilic inflammation and alveolar hemorrhage in the lungs but did not decrease viral infection, most likely because IP delivery could not achieve millimolar concentrations in the airways. These data show that thiol drugs inhibit SARS-CoV-2 infection in vitro and reduce SARS-CoV-2-related lung injury in vivo and provide strong rationale for trials of systemically delivered thiol drugs as COVID-19 treatments. We propose that antiviral effects of thiol drugs in vivo will require delivery directly to the airways to ensure millimolar drug concentrations and that thiol drugs with lower thiol pKa values are most likely to be effective.

A Midbrain Circuit that Mediates Headache Aversiveness in Rats.

Migraines are a major health burden, but treatment is limited because of inadequate understanding of neural mechanisms underlying headache. Imaging studies of migraine patients demonstrate changes in both pain-modulatory circuits and reward-processing regions, but whether these changes contribute to the experience of headache is unknown. Here, we demonstrate a direct connection between the ventrolateral periaqueductal gray (vlPAG) and the ventral tegmental area (VTA) that contributes to headache aversiveness in rats. Many VTA neurons receive monosynaptic input from the vlPAG, and cranial nociceptive input increases Fos expression in VTA-projecting vlPAG neurons. Activation of PAG inputs to the VTA induces avoidance behavior, while inactivation of these projections induces a place preference only in animals with headache. This work identifies a distinct pathway that mediates cranial nociceptive aversiveness.

Extracellular DNA, Neutrophil Extracellular Traps, and Inflammasome Activation in Severe Asthma.

Rationale: Extracellular DNA (eDNA) and neutrophil extracellular traps (NETs) are implicated in multiple inflammatory diseases. NETs mediate inflammasome activation and IL-1ß secretion from monocytes and cause airway epithelial cell injury, but the role of eDNA, NETs, and IL-1ß in asthma is uncertain. Objectives: To characterize the role of activated neutrophils in severe asthma through measurement of NETs and inflammasome activation. Methods: We measured sputum eDNA in induced sputum from 399 patients with asthma in the Severe Asthma Research Program-3 and in 94 healthy control subjects. We subdivided subjects with asthma into eDNA-low and -high subgroups to compare outcomes of asthma severity and of neutrophil and inflammasome activation. We also examined if NETs cause airway epithelial cell damage that can be prevented by DNase. Measurements and Main Results: We found that 13% of the Severe Asthma Research Program-3 cohort is "eDNA-high," as defined by sputum eDNA concentrations above the upper 95th percentile value in health. Compared with eDNA-low patients with asthma, eDNA-high patients had lower Asthma Control Test scores, frequent history of chronic mucus hypersecretion, and frequent use of oral corticosteroids for maintenance of asthma control (all P values <0.05). Sputum eDNA in asthma was associated with airway neutrophilic inflammation, increases in soluble NET components, and increases in caspase 1 activity and IL-1ß (all P values <0.001). In in vitro studies, NETs caused cytotoxicity in airway epithelial cells that was prevented by disruption of NETs with DNase. Conclusions: High extracellular DNA concentrations in sputum mark a subset of patients with more severe asthma who have NETs and markers of inflammasome activation in their airways.

Relative contributions and mapping of ventral tegmental area dopamine and GABA neurons by projection target in the rat.

The ventral tegmental area (VTA) is a heterogeneous midbrain structure that contains dopamine (DA), GABA, and glutamate neurons that project to many different brain regions. Here, we combined retrograde tracing with immunocytochemistry against tyrosine hydroxylase (TH) or glutamate decarboxylase (GAD) to systematically compare the proportion of dopaminergic and GABAergic VTA projections to 10 target nuclei: anterior cingulate, prelimbic, and infralimbic cortex; nucleus accumbens core, medial shell, and lateral shell; anterior and posterior basolateral amygdala; ventral pallidum; and periaqueductal gray. Overall, the non-dopaminergic component predominated VTA efferents, accounting for more than 50% of all projecting neurons to each region except the nucleus accumbens core. In addition, GABA neurons contributed no more than 20% to each projection, with the exception of the projection to the ventrolateral periaqueductal gray, where the GABAergic contribution approached 50%. Therefore, there is likely a significant glutamatergic component to many of the VTA's projections. We also found that VTA cell bodies retrogradely labeled from the various target brain regions had distinct distribution patterns within the VTA, including in the locations of DA and GABA neurons. Despite this patterned organization, VTA neurons comprising these different projections were intermingled and never limited to any one subregion. These anatomical results are consistent with the idea that VTA neurons participate in multiple distinct, parallel circuits that differentially contribute to motivation and reward. While attention has largely focused on VTA DA neurons, a better understanding of VTA subpopulations, especially the contribution of non-DA neurons to projections, will be critical for future work.

Trigeminocervical complex responses after lesioning dopaminergic A11 nucleus are modified by dopamine and serotonin mechanisms.

Both serotonergic and dopaminergic receptor modulation can alter trigeminal nociceptive processing, and descending A11 dopaminergic projections can affect trigeminal nociceptive transmission. Here we aimed to test the interaction between dopamine D(2) and serotonin 5-HT(1B/1D) receptors and their individual and combined effects in order to better understand the relationship of the descending influences of these systems on nociceptive trigeminovascular afferents. Extracellular recordings were made in the rat trigeminocervical complex in response to electrical stimulation of the dura mater and mechanical noxious and innocuous stimulation of the ipsilateral ophthalmic dermatome. The A11 nucleus was lesioned, and following the resultant facilitation of neuronal firing, one of a selective 5-HT(1B/1D) receptor agonist (naratriptan), selective 5-HT(1B/1D) receptor antagonist (GR127935), a selective D(2)-like receptor agonist (quinpirole), and a selective D(1)-like receptor agonist (dihydrexidine), or a combination of the above, were administered. Both quinpirole and quinpirole with naratriptan inhibited firing in the trigeminocervical complex evoked by noxious stimuli, reducing it below prelesion baseline, while the response to innocuous stimuli was reduced back to baseline. Both naratriptan alone, and quinpirole combined with GR127935, inhibited firing in the trigeminocervical complex evoked by noxious stimuli, returning it to prelesion baseline, while the response to innocuous stimuli remained facilitated. Immunohistochemical staining demonstrated D(2)-receptor and 5-HT(1B/1D)-receptor colocalization in the trigeminocervical complex. The data suggest that the serotonergic and dopaminergic antinociceptive pathways act simultaneously on neurons in the trigeminocervical complex, and both amine systems need to be functioning for trigeminal sensitization to be reversed.

Modulation of nocioceptive transmission with calcitonin gene-related peptide receptor antagonists in the thalamus.

Calcitonin gene-related peptide receptor antagonists are effective acute migraine treatments without the vascular contraindications associated with triptans. While it has been demonstrated that calcitonin gene-related peptide receptor antagonists act in the central nervous system, their effects in preclinical migraine models have been investigated in only the trigeminocervical complex. Migraine is a complex neurological disorder; sites in the brainstem and forebrain are clearly involved in its expression. We have performed electrophysiological recordings in thalamic neurons of rats responding to nocioceptive trigeminovascular inputs and tested the effect of olcegepant, a calcitonin gene-related peptide receptor antagonist (1 mg/kg, intravenously), on cell firing. We further tested the effect of microiontophoresed calcitonin gene-related peptide and the receptor antagonists calcitonin gene-related peptide 8-37 and olcegepant on thalamic cell firing, elicited by stimulation of the superior sagittal sinus or by microiontophoretic application of l-glutamate. Additionally, we used immunofluorescent staining to demonstrate the presence of functional calcitonin gene-related peptide receptors in the ventroposteromedial thalamic nucleus by specifically co-staining for the calcitonin gene-related peptide receptor subunits calcitonin receptor-like receptor and receptor activity modifying protein 1. Intravenously administered olcegepant significantly inhibited cell firing evoked by stimulation of the superior sagittal sinus as well as the background activity. Microiontophoresis of calcitonin gene-related peptide 8-37 also showed a significant inhibition of l-glutamate-evoked cell firing and firing evoked by stimulation of the superior sagittal sinus. Immunofluorescent staining confirmed the presence of the components of a functional calcitonin gene-related peptide receptor, the calcitonin receptor-like receptor and the receptor activity modifying protein 1, within the area of the ventroposteromedial thalamic nucleus. This is the first report on the efficacy of calcitonin gene-related peptide receptor antagonists at the level of third-order neurons in the migraine pathway, showing that the central effects of calcitonin gene-related peptide receptor antagonists extend beyond the trigeminocervical complex at least to the sensory thalamus.

Animal models of headache: from bedside to bench and back to bedside.

In recent years bench-based studies have greatly enhanced our understanding of headache pathophysiology, while facilitating the development of new headache medicines. At present, established animal models of headache utilize activation of pain-producing cranial structures, which for a complex syndrome, such as migraine, leaves many dimensions of the syndrome unstudied. The focus on modeling the central nociceptive mechanisms and the complexity of sensory phenomena that accompany migraine may offer new approaches for the development of new therapeutics. Given the complexity of the primary headaches, multiple approaches and techniques need to be employed. As an example, recently a model for trigeminal autonomic cephalalgias has been tested successfully, while by contrast, a satisfactory model of tension-type headache has been elusive. Moreover, although useful in many regards, migraine models are yet to provide a more complete picture of the disorder.

Dopamine: what's new in migraine?

PURPOSE OF REVIEW: Dopamine has been implicated in the pathophysiology of migraine, although its exact role remains unclear. Recent data offer some new perspective on a possible role for dopaminergic mechanisms in migraine. This review aims to summarize our current understanding of dopamine in migraine. RECENT FINDINGS: Direct application of dopamine and dopamine receptor agonists onto trigeminocervical complex neurons inhibits their activation after nociceptive stimulation. The dopaminergic A11 nucleus of the hypothalamus has been identified as the likely source of this dopamine. Recent evidence has shown that the genes for dopamine beta-hydroxylase and the dopamine transporter SLC6A3 may play a role in migraine pathophysiology, and dopamine has also been implicated in menstrual migraine. SUMMARY: Dopamine is currently considered to contribute to the pathophysiology of migraine, and dopamine receptor antagonists are prescribed in the treatment of acute migraine. Laboratory data suggest that the role of dopamine in migraine is more complex, perhaps due to the multiple receptors and levels of the brain involved in the disorder. These data suggest a reappraisal of dopaminergic therapeutic targets in migraine as our understanding of the role of this important biogenic amine is better characterized.

Neurons of the dopaminergic/calcitonin gene-related peptide A11 cell group modulate neuronal firing in the trigeminocervical complex: an electrophysiological and immunohistochemical study.

Activation of spinal trigeminal afferents innervating the cranial vasculature is likely to play a role in migraine, although some parts of the clinical presentation may have a dopaminergic basis. The A11 nucleus, located in the posterior hypothalamus, provides the only known source of descending dopaminergic innervation for the spinal gray matter. Extracellular recordings were made in the trigeminocervical complex (TCC) in response to electrical stimulation of the dura mater. Receptive fields were characterized by mechanical noxious and innocuous stimulation of the ipsilateral ophthalmic dermatome. Stimulation of the A11 significantly inhibited peri-middle meningeal artery dural and noxious pinch evoked firing of neurons in the TCC. This inhibition was reversed by the D(2) receptor antagonist eticlopride. Lesioning of the A11 significantly facilitated dural and noxious pinch and innocuous brush evoked firing from the TCC. In previous work using immunohistofluorescence, it was shown that D(1) and D(2) receptors were found in the rat TCC, and here we report, in addition, that D(4) and D(5) dopamine receptors are also present, whereas D(3) receptors are not. No dopamine receptors were present in the A11 nucleus itself. However, the A11 does contain dopamine and calcitonin gene-related peptide (CGRP) and, by this combination, is distinct from the neighboring CGRPergic subparafascicular nucleus. Exploration of dopaminergic influences and mechanisms in migraine may open up an almost untapped opportunity to pursue potential new therapeutic options for the disorder.

Comparison of the effects of central and peripheral dopamine receptor activation on evoked firing in the trigeminocervical complex.

Dopaminergic mechanisms have been suggested to play a role in migraine. Here, electrophysiological techniques were used to study the effects of intravenously administered centrally or peripherally active dopamine receptor agonists and antagonists on evoked firing in the trigeminocervical complex (TCC). After establishing baseline firing evoked by electrical stimulation of the dural middle meningeal artery (MMA) and mechanical noxious and innocuous stimulation of the ophthalmic dermatome, D(1)- or D(2)-like receptor agonists or antagonists were administered intravenously and the effect on firing was determined. In addition, with use of intravital microscopy, we monitored changes in dural vessel diameter in response to varying doses of D(1)- or D(2)-like receptor agonists to determine whether their effects were related to blood vessel caliber. The central D(2)-like receptor agonist quinpirole hydrochloride inhibited firing in the TCC evoked by stimulation of the MMA. Conversely, the central D(2)-like receptor antagonists, eticlopride hydrochloride and remoxipride hydrochloride, facilitated MMA-evoked firing and also firing evoked by noxious and innocuous stimulation of the ophthalmic dermatome. Both the peripheral D(1)-like receptor agonist fenoldopam and the central D(1)-like receptor agonists cis-(+/-)-1-(aminomethyl)-3,4-dihydro-3-phenyl-1H-2-benzopyran-5,6-diol hydrochloride (A68930 hydrochloride) and dihydrexidine facilitated innocuous brush-evoked firing, with A68930 hydrochloride having the greatest effect. The data suggest that dopamine binding to peripheral D(1)-like receptors may play a role in peripheral sensitization, and that the inhibitory or excitatory effects seen with administration of dopamine receptor agonists are independent of blood vessel changes. In addition, these studies maintain that central D(2)-like receptors inhibit trigeminocervical neurons, and may provide insight into the conflicting literature on the role of dopamine and its receptors in migraine.