The neuroimmune CGRP-RAMP1 axis tunes cutaneous adaptive immunity to the microbiota

The somatosensory nervous system surveils external stimuli at barrier tissues, regulating innate immune cells under infection and inflammation. The roles of sensory neurons in controlling the adaptive immune system, and more specifically immunity to the microbiota, however, remain elusive. Here, we identified a novel mechanism for direct neuroimmune communication between commensal-specific T lymphocytes and somatosensory neurons mediated by the neuropeptide calcitonin gene-related peptide (CGRP) in the skin. Intravital imaging revealed that commensal-specific T cells are in close proximity to cutaneous nerve fibers in vivo. Correspondingly, we observed upregulation of the receptor for the neuropeptide CGRP, RAMP1, in CD8+ T lymphocytes induced by skin commensal colonization. Neuroimmune CGRP-RAMP1 signaling axis functions in commensal-specific T cells to constrain Type 17 responses and moderate the activation status of microbiota-reactive lymphocytes at homeostasis. As such, modulation of neuroimmune CGRP-RAMP1 signaling in commensal-specific T cells shapes the overall activation status of the skin epithelium, thereby impacting the outcome of responses to insults such as wounding. The ability of somatosensory neurons to control adaptive immunity to the microbiota via the CGRP-RAMP1 axis underscores the various layers of regulation and multisystem coordination required for optimal microbiota-reactive T cell functions under steady state and pathology. Overall design: single cell RNA-seq: Skin CD8+ lymphocytes labeled with TotalSeqC hashtag antibodies (BioLegend) from S. epidermidis-associated mice (six Lck-Cre- Ramp1 fl/fl animals and six Lck Cre+ Ramp1 fl/fl animals) were sorted as DAPI- CD90.2+ TCR?+ CD4- CD8?+ TCR??- CD49f- NK1.1- B220- CD11b- CD11c- MHCII-, using a Sony MA900 cell sorter. All samples were pooled together, and approximately 15,000 sorted cells were loaded onto the Chromium Single Cell Controller (10X Genomics) to encapsulate cells into droplets. The library was prepared using a Chromium Single Cell 5' Reagent Kits v2 (10X Genomics) following manufacturer's instructions, and then sequenced on an Illumina Nextseq500 (Next Seq 500/550 High Output Kit v2, Illumina). ; bulk RNA-seq: CD4+ T cells from wild-type animals following S. epidermidis colonization were sorted into Th1 and Th17, processed and analyzed for RNA-seq as described in Linehan et al., 2018. Keratinocytes from the same Lck-Cre Ramp1 fl/fl experiment described above were sorted and processed for RNA-seq as described in Lima-Junior et al., 2021.