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BACKGROUND: The field of drug development is witnessing a remarkable surge in the development of innovative strategies. There is a need to develop technological platforms capable of generating human data prior to progressing to clinical trials. METHODS: Here we introduce a new flexible solution designed for the comprehensive monitoring of the natural human skin ecosystem's response to immunogenic drugs over time. Based on unique bioengineering to preserve surgical resections in a long survival state, it allows for the first time a comprehensive analysis of resident immune cells response at both organ and single-cell levels. RESULTS: Upon injection of the mRNA-1273 COVID-19 vaccine, we characterized precise sequential molecular events triggered upon detection of the exogenous substance. The vaccine consistently targets DC/macrophages and mast cells, regardless of the administration route, while promoting specific cell-cell communications in surrounding immune cell subsets. CONCLUSION: Given its direct translational relevance, this approach provides a multiscale vision of genuine human tissue immunity that could pave the way toward the development of new vaccination and drug development strategies.
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BACKGROUND: Mas-related G protein-coupled receptor X2 (MRGPRX2) is a promiscuous receptor on mast cells that mediates IgE-independent degranulation and has been implicated in multiple mast cell-mediated disorders, including chronic urticaria, atopic dermatitis, and pain disorders. Although it is a promising therapeutic target, few potent, selective, small molecule antagonists have been identified, and functional effects of human MRGPRX2 inhibition have not been evaluated in vivo. OBJECTIVE: We sought to identify and characterize novel, potent, and selective orally active small molecule MRGPRX2 antagonists for potential treatment of mast cell-mediated disease. METHODS: Antagonists were identified using multiple functional assays in cell lines overexpressing human MRGPRX2, LAD2 mast cells, human peripheral stem cell-derived mast cells, and isolated skin mast cells. Skin mast cell degranulation was evaluated in Mrgprb2em(-/-) knockout and Mrgprb2em(MRGPRX2) transgenic human MRGPRX2 knock-in mice by assessment of agonist-induced skin vascular permeability. Ex vivo skin mast cell degranulation and associated histamine release was evaluated by microdialysis of human skin tissue samples. RESULTS: MRGPRX2 antagonists potently inhibited agonist-induced MRGPRX2 activation and mast cell degranulation in all mast cell types tested in an IgE-independent manner. Orally administered MRGPRX2 antagonists also inhibited agonist-induced degranulation and resulting vascular permeability in MRGPRX2 knock-in mice. In addition, antagonist treatment dose dependently inhibited agonist-induced degranulation in ex vivo human skin. CONCLUSIONS: MRGPRX2 small molecule antagonists potently inhibited agonist-induced mast cell degranulation in vitro and in vivo as well as ex vivo in human skin, supporting potential therapeutic utility as a novel treatment for multiple human diseases involving clinically relevant mast cell activation.
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FLG is a well-known biomarker of atopic dermatitis and skin dryness. Its full proteolysis (or filaggrinolysis) produces the major constituents of the natural moisturizing factor. Some proteases/peptidases remain to be identified in this multistep process. Mining 16 omics analyses, we identified prolyl endopeptidase (PREP) as a candidate peptidase. Indirect immunofluorescence and confocal analysis demonstrated its localization in the granular and deep cornified layers, where it colocalized with FLG. Tandem mass spectroscopy and fluorescent quenching activity assays showed that PREP cleaved several synthetic peptides derived from the FLG sequence, at the carboxyl side of an internal proline. Deimination of these peptides increased PREP enzymatic efficiency. Specific inhibition of PREP in reconstructed human epidermis using benzyloxycarbonyl-pro-prolinal induced the accumulation of FLG monomers. Downregulation of PREP expression in reconstructed human epidermis using RNA interference confirmed the impact of PREP on FLG metabolism and highlighted a more general role of PREP in keratinocyte differentiation. Indeed, quantitative global proteomic, western blotting, and RT-qPCR analyses showed a strong reduction in the expression of bleomycin hydrolase, known to be involved in filaggrinolysis, and of several other actors of cornification such as loricrin. Consequently, at the functional level, the transepidermal electric resistance was drastically reduced.
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BACKGROUND: Systemic mastocytosis (SM) is a clonal disorder of mast cells (MCs) frequently associated with vertebral osteoporosis (OP) and subsequent vertebral fractures (VFs). The natural history of this OP remains unclear. Importantly, we do not know whether OP represents an early event triggered alongside MC abnormalities, and whether MC clonality is sufficient to trigger osteoporosis. OBJECTIVE: To describe OP in patients with medullar clonality in cutaneous mastocytosis (CM) and monoclonal mast cell activation syndrome (MMAS) and to compare their osteoporosis characteristics with those of nonadvanced SM patients (bone marrow mastocytosis and indolent systemic mastocytosis). METHODS: We retrospectively analyzed clinical, biological, and densitometric data of 27 CM, 13 MMAS, and 135 SM patients from the Mastocytosis Expert Center (CEREMAST) in Toulouse, France. RESULTS: The OP (respectively 3.7, 30.8, and 34.1%) and VFs (0.0%, 15.4%, and 20%) were less frequent in CM than in MMAS and SM, despite the presence of clonal MCs in the bone marrow. Most patients with OP and VFs in the non-SM groups had the usual risk factors for OP. Interestingly, the only non-SM patient with a typical SM-like OP had high bone marrow tryptase, developed bone marrow KIT mutation during follow-up, and had a family history of SM. Our data show that OP is not a common clinical finding in CM but is frequent in MMAS. When OP and VFs occur in CM and MMAS patients, they differ from the usual phenotype of SM bone fragility. CONCLUSIONS: Our findings suggest that, in most CM patients, the meaning and management of OP differs from that of OP in MMAS and nonadvanced SM. Prospective longitudinal studies and the validation of predictors are needed to identify CM and MMAS patients developing SM-related OP.