Neutrophil trapping and nexocytosis, mast cell-mediated processes for inflammatory signal relay.

Neutrophils are sentinel immune cells with essential roles for antimicrobial defense. Most of our knowledge on neutrophil tissue navigation derived from wounding and infection models, whereas allergic conditions remained largely neglected. Here, we analyzed allergen-challenged mouse tissues and discovered that degranulating mast cells (MCs) trap living neutrophils inside them. MCs release the attractant leukotriene B4 to re-route neutrophils toward them, thus exploiting a chemotactic system that neutrophils normally use for intercellular communication. After MC intracellular trap (MIT) formation, neutrophils die, but their undigested material remains inside MC vacuoles over days. MCs benefit from MIT formation, increasing their functional and metabolic fitness. Additionally, they are more pro-inflammatory and can exocytose active neutrophilic compounds with a time delay (nexocytosis), eliciting a type 1 interferon response in surrounding macrophages. Together, our study highlights neutrophil trapping and nexocytosis as MC-mediated processes, which may relay neutrophilic features over the course of chronic allergic inflammation.

Robust and Objective Evaluation of Superficial Punctate Keratopathy in a Murine Dry Eye Model.

Purpose: To establish a robust and objective method to evaluate (SPK) superficial punctate keratopathy in a murine dry eye model by developing a reliable photographic system. Design: Experimental study. Subjects: A murine dry eye model was generated by exorbital lacrimal gland excision. Sham-operated mice were used as healthy controls. For the sham operation, an incision was made without touching the gland. Methods: A photographic system was constructed, consisting of an LED lamp and a digital camera fitted with a zoom lens and sharp cut filter. SPK was detected by applying fluorescein solution. To validate the system, SPK was compared between dry eye mice and healthy control mice, and diquafosol (DIQUAS ophthalmic solution 3%; Santen Pharmaceutical Co., Ltd.) or cyclosporine (PAPILOCK Mini ophthalmic solution 0.1%; Santen Pharmaceutical Co., Ltd.) was used to dry eye mice. Main Outcome Measures: SPK was evaluated using the parameters of fluorescence score and fluorescein-stained area. Results: The photographs clearly indicated SPK in dry eye mice. A fluorescence score of 0 to 9 could be easily assessed, and the fluorescein-stained area was quantifiable. The fluorescein-stained area correlated with fluorescence score (correlation coefficient: 0.98), with good interobserver reliability (intraclass correlation coefficient: 0.999). The fluorescein-stained area increased significantly in dry eye mice compared with that of healthy controls (P < 0.0001). Both types of therapeutic eye drops decreased the fluorescein-stained area relative to saline-treated mice (P < 0.05 in diquafosol vs. saline; P < 0.01 in cyclosporine vs. saline). Conclusions: This newly developed system is a robust alternative for quantitative evaluation of SPK in a murine dry eye model. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Development of sexual dimorphism of skeletal muscles through the adrenal cortex, caused by androgen-induced global gene suppression.

The zona fasciculata (zF) in the adrenal cortex contributes to multiple physiological actions through glucocorticoid synthesis. The size, proliferation, and glucocorticoid synthesis characteristics are all female biased, and sexual dimorphism is established by androgen. In this study, transcriptomes were obtained to unveil the sex differentiation mechanism. Interestingly, both the amount of mRNA and the expressions of nearly all genes were higher in females. The expression of Nr5a1, which is essential for steroidogenic cell differentiation, was also female biased. Whole-genome studies demonstrated that NR5A1 regulates nearly all gene expression directly or indirectly. This suggests that androgen-induced global gene suppression is potentially mediated by NR5A1. Using Nr5a1 heterozygous mice, whose adrenal cortex is smaller than the wild type, we demonstrated that the size of skeletal muscles is possibly regulated by glucocorticoid synthesized by zF. Taken together, considering the ubiquitous presence of glucocorticoid receptors, our findings provide a pathway for sex differentiation through glucocorticoid synthesis.

Bicarbonate signalling via G protein-coupled receptor regulates ischaemia-reperfusion injury.

Homoeostatic regulation of the acid-base balance is essential for cellular functional integrity. However, little is known about the molecular mechanism through which the acid-base balance regulates cellular responses. Here, we report that bicarbonate ions activate a G protein-coupled receptor (GPCR), i.e., GPR30, which leads to Gq-coupled calcium responses. Gpr30-Venus knock-in mice reveal predominant expression of GPR30 in brain mural cells. Primary culture and fresh isolation of brain mural cells demonstrate bicarbonate-induced, GPR30-dependent calcium responses. GPR30-deficient male mice are protected against ischemia-reperfusion injury by a rapid blood flow recovery. Collectively, we identify a bicarbonate-sensing GPCR in brain mural cells that regulates blood flow and ischemia-reperfusion injury. Our results provide a perspective on the modulation of GPR30 signalling in the development of innovative therapies for ischaemic stroke. Moreover, our findings provide perspectives on acid/base sensing GPCRs, concomitantly modulating cellular responses depending on fluctuating ion concentrations under the acid-base homoeostasis.