Prior studies have demonstrated that the ion channel transient receptor potential vanilloid 4 (TRPV4) is functionally expressed in airway smooth muscle cells and that TRPV4 single nucleotide polymorphisms are associated with airflow obstruction in patients with chronic obstructive pulmonary disease. We sought to use isometric tension measurements in ex vivo airways to determine whether short-term pharmacological activation of TRPV4 with thewould constrict human bronchial tissue. As predicted, transient receptor potential vanilloid 4 activation in the human airway produces contractions that are blocked by the nonselective transient receptor potential channel blocker ruthenium red. Moreover, the novel TRPV4-selective blocker GSK2334775 [(R)-6-(methylsulfonyl)-3-((4-(pyrrolidin-1-yl)piperindin-1-yl)methyl)-N-(2,2,2,-trifluoro-1-phenylethyl)-2-(3-(trifluoromethyl)phenyl)quinoline-4-carboxamide] inhibited these contractions over a concentration range consistent with its in vitro potency against recombinant and native TRPV4-containing channels. Surprisingly, TRPV4-dependent contractions were also blocked by a 5-lipoxygenase inhibitor and two structurally distinct cysteinyl leukotriene 1 receptor antagonists. In aggregate, our results fail to support the hypothesis that TRPV4 in airway smooth muscle cells regulates airway contractility short term. Rather, we provide pharmacological evidence that TRPV4 activation causes human airway constriction that is entirely dependent upon the production of cysteinyl leukotrienes. Together, these data identify a novel mechanism by which TRPV4 activation may contribute to pathologic remodeling and inflammation, in addition to airflow obstruction, in the diseased human respiratory tract.
CXC chemokine receptor 2 (CXCR2) is a key receptor in the chemotaxis of neutrophils to sites of inflammation. The studies reported here describe the pharmacological characterization of danirixin, a CXCR2 antagonist in the diaryl urea chemical class. Danirixin has high affinity for CXCR2, with a negative log of the 50% inhibitory concentration (pIC) of 7.9 for binding to Chinese hamster ovary cell (CHO)-expressed human CXCR2, and 78-fold selectivity over binding to CHO-expressed CXCR1. Danirixin is a competitive antagonist against CXCL8 in Ca-mobilization assays, with a K (the concentration of antagonist that binds 50% of the receptor population) of 6.5 nM and antagonist potency (pA) of 8.44, and is fully reversible in washout experiments over 180 minutes. In rat and human whole-blood studies assessing neutrophil activation by surface CD11b expression following CXCL2 (rat) or CXCL1 (human) challenge, danirixin blocks the CD11b upregulation with pICs of 6.05 and 6.3, respectively. Danirixin dosed orally also blocked the influx of neutrophils into the lung in vivo in rats following aerosol lipopolysaccharide or ozone challenge, with median effective doses (EDs) of 1.4 and 16 mg/kg respectively. Thus, danirixin would be expected to block chemotaxis in disease states in which neutrophils are increased in response to inflammation, such as pulmonary diseases. In comparison with navarixin, a CXCR2 antagonist from a different chemical class, the binding characterization of danirixin is distinct. These observations may offer insight into the previously observed clinical differences in induction of neutropenia between these compounds.
Neuromodulation of immune function by stimulating the autonomic connections to the spleen has been demonstrated in rodent models. Consequently, neuroimmune modulation has been proposed as a new therapeutic strategy for the treatment of inflammatory conditions. However, demonstration of the translation of these immunomodulatory mechanisms in anatomically and physiologically relevant models is still lacking. Additionally, translational models are required to identify stimulation parameters that can be transferred to clinical applications of bioelectronic medicines. Here, we performed neuroanatomical and functional comparison of the mouse, rat, pig, and human splenic nerve using in vivo and ex vivo preparations. The pig was identified as a more suitable model of the human splenic innervation. Using functional electrophysiology, we developed a clinically relevant marker of splenic nerve engagement through stimulation-dependent reversible reduction in local blood flow. Translation of immunomodulatory mechanisms were then assessed using pig splenocytes and two models of acute inflammation in anesthetized pigs. The pig splenic nerve was shown to locally release noradrenaline upon stimulation, which was able to modulate cytokine production by pig splenocytes. Splenic nerve stimulation was found to promote cardiovascular protection as well as cytokine modulation in a high- and a low-dose lipopolysaccharide model, respectively. Importantly, splenic nerve–induced cytokine modulation was reproduced by stimulating the efferent trunk of the cervical vagus nerve. This work demonstrates that immune responses can be modulated by stimulation of spleen-targeted autonomic nerves in translational species and identifies splenic nerve stimulation parameters and biomarkers that are directly applicable to humans due to anatomical and electrophysiological similarities.
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