Local Modulation of Lymph Node Stroma by Sensory Neurons II

Immune responses within barrier tissues are regulated, in part, by nociceptors, specialized peripheral sensory neurons that detect noxious stimuli. Previous work has shown that nociceptor ablation not only alters local responses at peripheral sites of immune challenge, but also within draining lymph nodes (LNs). The mechanism and significance of nociceptor-dependent modulation of LN homeostasis are unknown. Indeed, although sympathetic innervation of LNs is well documented, it has been unclear whether the LN parenchyma itself is innervated by sensory neurons. Here, using a combination of high-resolution imaging, retrograde viral tracing, optogenetics, and single-cell transcriptomics (scRNA-seq), we describe a sensory neuro-immune circuit that is preferentially located in the outermost cortex of skin-draining LNs. Transcriptomic profiling revealed that sensory neurons that innervate LNs are composed of at least four discrete subsets with a predominance of peptidergic nociceptors, an innervation pattern that is distinct from that in the surrounding skin. To identify potential LN-resident communication partners for LN-innervating sensory neurons, we employed scRNA-seq to generate an atlas of all murine LN cells and, based on receptor-ligand expression patterns, nominated candidate target populations among stromal and immune cells. We experimentally validated these inferred connections by comparing scRNA-seq signatures before and after selective optogenetic stimulation of LN-innervating axons. Acute neuronal activation triggered rapid transcriptional changes preferentially in endothelium and other nodal stroma cells, as well as in several innate leukocyte populations. Thus, LNs are monitored by a unique population of sensory neurons that possess profound immunomodulatory potential. Left and Right Inguinal LNs from 7 mice were digested using a modified protocol for gentle cellularization (Fletcher et al. Front Immunol 2011). A portion of cells were retained for Seq-Well scRNA-seq representing the "whole" or un-depleted fraction, the remaining were depleted of CD3e and CD19 expressing cells using the Miltenyi microbead kit, and run on Seq-Well arrays in parallel. To specifically activate LN-innervating neurons in vivo, 4 of the 7 mice were Nav1.8Cre/+ x Rosa26ChR2-eYFP/+ (ChR2+) in which Nav1.8 lineage neurons expressed ChR2. The Left iLN-innervating sensory neurons were selectively activated with blue light (473 nm) directed through an optical fiber (200 µm tip diameter) towards a region of the subiliac artery adjacent to the hilus, the predominant site where sensory nerve bundles enter the iLN. A control cohort of 3 Nav1.8Cre/+ x Rosa26eYFP/+ (ChR2-) animals, which expressed eYFP instead of ChR2 in Nav1.8 lineage neurons, were subjected to identical photostimulation, dissociation, cellular enrichment, and Seq-Well analysis.