Dermal TRPV1 innervations engage a macrophage and fibroblast containing pathway to activate hair growth [dataset2]

Pain, detected by nociceptors, is an integral part of injury, yet whether and how it can impact tissue physiology and recovery remain understudied. Here we applied chemogenetics in mice to locally activate dermal TRPV1 innervations in naïve skin and found it triggered new regenerative cycling by dormant hair follicles (HFs).This was preceded by rapid apoptosis of dermal macrophages, mediated by the neuropeptide calcitonin gene-related peptide (CGRP). TRPV1 activation also triggered a macrophage-dependent induction of Spp1-expressing dermal fibroblasts. The neuropeptide CGRP andSpp1were essential for the nociceptor-triggered hair growth. Finally, we show that epidermal abrasion injury induced Spp1-expressing dermal fibroblasts and hair growth via a TRPV1 neuron and CGRP dependent mechanism.Collectively, these data demonstrate a role for TRPV1 nociceptors in orchestrating a macrophage and fibroblast-supported mechanism to promote hair growth, and enabling the efficient restoration of this mechano- and thermo-protective barrier after wounding. Overall design: Back skin from TRPV1hM3Dq and control mice (three mice per group) treated daily with a CNO intradermal injection for three consecutive days, were dissected, separated into the subcutis and dermal-epidermal fractions and then digested. The dermal-epidermal and subcutis fractions were stained separately with TotalSeq™ anti-mouse Hashtag antibodies (TotalSeq™ A0301 and A0302 from BioLegend) to mark the cells' anatomical location. Thy1+ SCA1+ stromal cells (CD45-CD31- EpCAM-) were sorted and analysed. Treatment groups (two) were run on separate lanes of a 10X Chromium chip with 3' v.2 chemistry (10X Genomics) as per the manufacturer's instructions by the UCSF Institute for Human Genetics Sequencing Core. Transcripts captured in all the cells encapsulated with a bead were uniquely barcoded using a combination of a 16 bp 10x Barcode and a 10 bp unique molecular identifier (UMI). cDNA libraries were generated using the Chromium™ Single Cell 3' Library & Gel Bead Kit v3 (10x Genomics) following the detailed protocol provided by the manufacturer. Libraries were sequenced with the NovaSeq 6000 platform (S1 Cartridge, Illumina) in 150 bp paired-end mode. The hashtag library was demultiplexed using CellRanger software (version 7.0.0), discriminating between spliced and unspliced transcripts. Aligned spliced reads were used to quantify the expression level of mouse genes and generation of gene-barcode matrix. Subsequent data analysis was performed using Seurat R package (4.0.4) and Scanpy (1.8.2). Quality control was performed, and viable cells were selected by excluding cells with features lower than 200 and above 4000, as well as cells having more than 5% of mitochondrial transcripts. Dermal and epidermal derived cells were demultiplexed with the HTODemux function integrated in Seurat with standard settings and dermal derived cells were included in the analysis all doublets were removed for the analysis. 2000 most variable genes used for the anchoring process were used for downstream analysis to calculate principal components, after log-normalization and scaling. HTO; Barcode sequence; Hashtagged sample Hashtag_1_(A0301); ACCCACCAGTAAGAC; epidermis+dermis Hashtag_2_(A0302); GGTCGAGAGCATTCA; subcutis