Mast cell activation disrupts interactions between endothelial cells and pericytes during early life allergic asthma.

Allergic asthma generally starts during early life and is linked to substantial tissue remodeling and lung dysfunction. Although angiogenesis is a feature of the disrupted airway, the impact of allergic asthma on the pulmonary microcirculation during early life is unknown. Here, using quantitative imaging in precision-cut lung slices (PCLSs), we report that exposure of neonatal mice to house dust mite (HDM) extract disrupts endothelial cell/pericyte interactions in adventitial areas. Central to the blood vessel structure, the loss of pericyte coverage was driven by mast cell (MC) proteases, such as tryptase, that can induce pericyte retraction and loss of the critical adhesion molecule N-cadherin. Furthermore, spatial transcriptomics of pediatric asthmatic endobronchial biopsies suggests intense vascular stress and remodeling linked with increased expression of MC activation pathways in regions enriched in blood vessels. These data provide previously unappreciated insights into the pathophysiology of allergic asthma with potential long-term vascular defects.

Neutrophil breaching of the blood vessel pericyte layer during diapedesis requires mast cell-derived IL-17A.

Neutrophil diapedesis is an immediate step following infections and injury and is driven by complex interactions between leukocytes and various components of the blood vessel wall. Here, we show that perivascular mast cells (MC) are key regulators of neutrophil behaviour within the sub-endothelial space of inflamed venules. Using confocal intravital microscopy, we observe directed abluminal neutrophil motility along pericyte processes towards perivascular MCs, a response that created neutrophil extravasation hotspots. Conversely, MC-deficiency and pharmacological or genetic blockade of IL-17A leads to impaired neutrophil sub-endothelial migration and breaching of the pericyte layer. Mechanistically, identifying MCs as a significant cellular source of IL-17A, we establish that MC-derived IL-17A regulates the enrichment of key effector molecules ICAM-1 and CXCL1 in nearby pericytes. Collectively, we identify a novel MC-IL-17A-pericyte axis as modulator of the final steps of neutrophil diapedesis, with potential translational implications for inflammatory disorders driven by increased neutrophil diapedesis.

Age-related changes in the local milieu of inflamed tissues cause aberrant neutrophil trafficking and subsequent remote organ damage.

Aging is associated with dysregulated immune functions. Here, we investigated the impact of age on neutrophil diapedesis. Using confocal intravital microscopy, we found that in aged mice, neutrophils adhered to vascular endothelium in inflamed tissues but exhibited a high frequency of reverse transendothelial migration (rTEM). This retrograde breaching of the endothelium by neutrophils was governed by enhanced production of the chemokine CXCL1 from mast cells that localized at endothelial cell (EC) junctions. Increased EC expression of the atypical chemokine receptor 1 (ACKR1) supported this pro-inflammatory milieu in aged venules. Accumulation of CXCL1 caused desensitization of the chemokine receptor CXCR2 on neutrophils and loss of neutrophil directional motility within EC junctions. Fluorescent tracking revealed that in aged mice, neutrophils undergoing rTEM re-entered the circulation and disseminated to the lungs where they caused vascular leakage. Thus, neutrophils stemming from a local inflammatory site contribute to remote organ damage, with implication to the dysregulated systemic inflammation associated with aging.

Local microvascular leakage promotes trafficking of activated neutrophils to remote organs.

Increased microvascular permeability to plasma proteins and neutrophil emigration are hallmarks of innate immunity and key features of numerous inflammatory disorders. Although neutrophils can promote microvascular leakage, the impact of vascular permeability on neutrophil trafficking is unknown. Here, through the application of confocal intravital microscopy, we report that vascular permeability-enhancing stimuli caused a significant frequency of neutrophil reverse transendothelial cell migration (rTEM). Furthermore, mice with a selective defect in microvascular permeability enhancement (VEC-Y685F-ki) showed reduced incidence of neutrophil rTEM. Mechanistically, elevated vascular leakage promoted movement of interstitial chemokines into the bloodstream, a response that supported abluminal-to-luminal neutrophil TEM. Through development of an in vivo cell labeling method we provide direct evidence for the systemic dissemination of rTEM neutrophils, and showed them to exhibit an activated phenotype and be capable of trafficking to the lungs where their presence was aligned with regions of vascular injury. Collectively, we demonstrate that increased microvascular leakage reverses the localization of directional cues across venular walls, thus causing neutrophils engaged in diapedesis to reenter the systemic circulation. This cascade of events offers a mechanism to explain how local tissue inflammation and vascular permeability can induce downstream pathological effects in remote organs, most notably in the lungs.

The Mast Cell Antibody-Dependent Degranulatory Synapse.

Mast cells are key effector cells in inflammation that can be activated by specific antigens via IgE or IgG binding on their FcR. Aggregation of mast cell Fc receptors by cell-bound antigens induces mast cell polarized degranulation toward the stimulatory cell, a process named antibody-dependent degranulatory synapse (ADDS). This polarized degranulation allows mast cells to expose bioactive material embedded in the granule matrix toward the antibody-targeted cell and is accompanied by the formation of a signaling area at the cell-cell contact site. In this chapter, we describe (1) how to stimulate mast cells with cell-bound antigens and (2) how to monitor ADDS formation and to investigate ADDS characteristics by confocal microscopy.

IL-33 fine tunes mast cell degranulation and chemokine production at the single-cell level.

BACKGROUND: Mast cells are versatile key components of allergy and inflammation known to respond to both innate and adaptive immunologic stimuli. However, the response of individual mast cells to cumulative stimuli remains poorly understood. OBJECTIVES: We sought to dissect mast cell responses at the single-cell level and their potentiation by IL-33. METHODS: We monitored mast cell degranulation in real time by exploiting the capacity of fluorochrome-labeled avidin to stain degranulating cells. During the degranulation process, the granule matrix is externalized and immediately bound by fluorochrome-labeled avidin present in the culture medium. The degranulation process is monitored by using either time-lapse microscopy or fluorescence-activated cell sorting analysis. RESULTS: Single-cell analysis revealed a strong heterogeneity of individual mast cell degranulation responses. We observed that the number of degranulating mast cells was graded according to the FcεRI stimulation strength, whereas the magnitude of individual mast cell degranulation remained unchanged, suggesting an all-or-none response of mast cells after FcεRI triggering. IL-33 pretreatment increased not only the number of degranulating and chemokine-producing mast cells but also the magnitude of individual mast cell degranulation and chemokine production. CONCLUSION: We illustrate the effect of IL-33 on mast cell biology at the single-cell level by showing that IL-33 potentiates IgE-mediated mast cell responses by both increasing the number of responding cells and enhancing the responses of individual mast cells.

Mast cells form antibody-dependent degranulatory synapse for dedicated secretion and defence.

Mast cells are tissue-resident immune cells that play a key role in inflammation and allergy. Here we show that interaction of mast cells with antibody-targeted cells induces the polarized exocytosis of their granules resulting in a sustained exposure of effector enzymes, such as tryptase and chymase, at the cell-cell contact site. This previously unidentified mast cell effector mechanism, which we name the antibody-dependent degranulatory synapse (ADDS), is triggered by both IgE- and IgG-targeted cells. ADDSs take place within an area of cortical actin cytoskeleton clearance in the absence of microtubule organizing centre and Golgi apparatus repositioning towards the stimulating cell. Remarkably, IgG-mediated degranulatory synapses also occur upon contact with opsonized Toxoplasma gondii tachyzoites resulting in tryptase-dependent parasite death. Our results broaden current views of mast cell degranulation by revealing that human mast cells form degranulatory synapses with antibody-targeted cells and pathogens for dedicated secretion and defence.