Project description:Background: Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by eczematous lesions and intense pruritus. Skin inflammation mediated by transient receptor potential vanillin 3 (TRPV3) in keratinocytes is a key mechanism driving the progression of AD. Paeonol is a bioactive phenolic compound extracted from the root bark of the Paeonia genus, which has demonstrated anti-inflammatory activities. Whether paeonol alleviates skin inflammation by inhibiting TRPV3 in the treatment of AD remains unclear.Objective: This study aims to investigate the therapeutic efficacy of paeonol against AD and its underlying anti-inflammatory mechanism.Methods: The therapeutic effects of paeonol were evaluated in a mouse model of AD induced by MC903 or carvacrol, as well as in tumor necrosis factor-alpha (TNF-α)-stimulated HaCaT cell line. Itch-related behaviors, hematoxylin-eosin (H&E) staining and toluidine blue staining were assessed in AD mice to evaluate the anti-atopic activity of paeonol. TRPV3 expression and function in keratinocytes were examined by Quantitative Real-time-PCR (qPCR), WB, immunofluorescence (IF) and Ca2+ imaging experiments. The anti-inflammation effect and its mechanisms of paeonol were measured by RNA sequencing (RNA-seq), H&E staining, qPCR and ELISA. Whole-cell patch-clamp were used to measure the excitability of trigeminal ganglion (TG) neurons treated by Chemokine (C-C motif) ligand 3 (CCL3).Results: Paeonol significantly alleviated MC903 and carvacrol-induced pruritus, epidermal hyperplasia and skin inflammation. Paeonol inhibited the expression and function of TRPV3 in keratinocytes, thereby inhibiting the activating of nuclear factor κB (NF-κB) pathway and reducing the secretion of CCL3. Downregulation of CCL3 expression in keratinocytes reduces the number of macrophages in the skin and inhibits their polarization to the M1 type. Moreover, paeonol also reversed the hyperexcitability of TG induced by increased CCL3, thus alleviated pruritus. Conclusion: Our study demonstrated that the activation of TRPV3 in keratinocytes aggravated skin inflammation and pruritus by promoting the secretion of CCL3. Paeonol alleviated AD by inhibiting the TRPV3/NF-κB/CCL3 axis in keratinocytes, thereby reducing macrophage-related skin inflammation, and inhibiting the hyperexcitability of TG neurons. These findings provide a novel therapeutic mechanism of paeonol in treating AD.

Project description:Background: Anti-type 2 cytokine therapies represent promising interventions for chronic itch; however, their precise mechanisms in restoring nerve architecture and mitigating inflammation and pruritus remain incompletely understood. This study aimed to elucidate the mechanistic roles of IL-4, IL-13, and IL-31 in the pathophysiology of itch associated with type 2 inflammatory skin diseases. Methods: The effect of IL-4, IL-13, and/or IL-31 on neurite outgrowth and/or transcriptomic changes were analyzed in human and mouse dorsal root ganglion (DRG) neuronal cultures. Mouse ear pinnae were processed for histologic, transcriptomic, and proteomic analyses 4 days after intradermal injection of IL-4, IL-13, and/or IL-31. To evaluate functional correlations with neuronal responses, mice were subcutaneously challenged with IL-4, IL-13, and/or IL-31, and scratching behavior was monitored. Association between IL-4/IL-13–IL-4Rα axis and severity of atopic dermatitis (AD) was evaluated through correlative analyses of human DRG transcriptomic changes and AD transcriptomic datasets (GSE130588 and BioMap consortium). Results: IL-4 and IL-13 promote mouse and human DRG sensory neuron growth, with effects similar to or greater than IL-31. In mice, intradermal IL-4, IL-13, and IL-31 increased epidermal nerve growth; however, only IL-4 and IL-13 induced hyperplasia and immune cell recruitment. Multi-omic analyses revealed that IL-4 and IL-13 have a broader impact on neuroimmune interactions than IL-31. In a murine DRG neuron-eosinophil co-culture, IL-4Rα blockade reduced neurite growth. IL-13 and IL-31 elicited acute scratching, demonstrating their roles as direct pruritogens; IL-4 synergistically enhanced IL-13-induced itch, resulting in greater pruritic responses than IL-31. Additionally, a set of itch-associated genes upregulated by IL-4 and IL-13 and downregulated by dupilumab-mediated IL-4Rα blockade in human DRG neuronal cultures showed positive correlation with AD severity. Conclusions: These findings establish the IL-4/IL-13–IL-4Rα axis as a key regulator of inflammatory skin nerve innervation, neuroimmune interactions, barrier integrity, and itch response, highlighting its mechanistic role in modulating sensory neuronal function and shaping the inflammatory microenvironment that drives itch pathophysiology.

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Multi-omics Reveals CCL3 Driving Neuronal Sensitization in Chronic Pruritus of Unknown Origin

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