Dermal TRPV1 innervations engage a macrophage and fibroblast containing pathway to activate hair growth [dataset1]

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 expressing and control mice (two mice per group) were collected 3.5 hours after a single intradermal CNO injection. The subcutis was removed mechanically and the dermal-epidermal fractions were then digested. Then, control and TRPV1 activated samples were stained using viability dyw and CD45 antibody and sorted. 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_(B0301); ACCCACCAGTAAGAC; mouse1 Hashtag_2_(B0302); GGTCGAGAGCATTCA; mouse2 Hashtag_3_(B0303); CTTGCCGCATGTCAT; mouse3 Hashtag_3_(B0303); CTTGCCGCATGTCAT; mouse4

痛觉由伤害性感受器（nociceptors）感知，是损伤过程中不可或缺的组成部分，但痛觉是否以及如何影响组织生理与修复，目前仍有待深入研究。本研究在小鼠体内应用化学遗传学手段，于未受损伤的皮肤中局部激活皮肤TRPV1神经支配，发现其可触发休眠毛囊（hair follicles, HFs）启动新一轮再生周期。该过程的上游事件为：神经肽降钙素基因相关肽（calcitonin gene-related peptide, CGRP）介导了皮肤巨噬细胞的快速凋亡。TRPV1激活还可触发巨噬细胞依赖性的、表达Spp1的皮肤成纤维细胞的诱导生成。神经肽CGRP与Spp1对于伤害性感受器触发的毛囊生长至关重要。最后，本研究证实表皮擦伤损伤可通过TRPV1神经元及CGRP依赖性机制，诱导表达Spp1的皮肤成纤维细胞生成并促进毛囊生长。综上，本研究数据表明，TRPV1伤害性感受器可通过调控巨噬细胞与成纤维细胞协同的信号通路，促进毛囊生长，从而在皮肤损伤后高效恢复其机械与热防护屏障功能。

整体实验设计：向表达TRPV1-hM3Dq的小鼠及对照小鼠（每组2只）单次皮内注射CNO 3.5小时后，采集背部皮肤。先通过机械方法去除皮下组织，随后消化分离真皮-表皮组分。随后，对照与TRPV1激活组样本分别用活力染料与CD45抗体染色并进行分选。两组处理样本按照制造商操作流程，由加州大学旧金山分校人类遗传学研究所测序核心实验室使用10X Chromium芯片搭配3' v2化学试剂（10X Genomics）进行单细胞测序。包裹在磁珠中的所有细胞的转录本，通过16bp的10x条形码与10bp的唯一分子标识符（unique molecular identifier, UMI）的组合进行唯一标记。按照制造商提供的详细方案，使用Chromium™单细胞3'文库与磁珠试剂盒v3（10X Genomics）构建cDNA文库。使用Illumina NovaSeq 6000平台（S1流动槽）以150bp双端测序模式对文库进行测序。使用CellRanger软件（版本7.0.0）对标签文库进行解多路复用，区分剪接与未剪接转录本。使用比对后的剪接reads定量小鼠基因的表达水平，并生成基因-条形码矩阵。后续数据分析使用Seurat R包（4.0.4）与Scanpy（1.8.2）完成。质量控制步骤中，我们剔除了基因数低于200、高于4000，以及线粒体转录本占比超过5%的细胞，以筛选存活细胞。使用Seurat内置的HTODemux函数（默认参数）对真皮与表皮来源的细胞进行解多路复用，仅保留真皮来源细胞用于后续分析，并移除所有双细胞。在对数归一化与缩放后，选取前2000个变异度最高的基因用于锚定分析，并以此计算主成分。

HTO：条形码序列；带标签样本：
Hashtag_1_(B0301)：ACCCACCAGTAAGAC，对应小鼠1
Hashtag_2_(B0302)：GGTCGAGAGCATTCA，对应小鼠2
Hashtag_3_(B0303)：CTTGCCGCATGTCAT，对应小鼠3、小鼠4