Project description: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.
Project description: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.
Project description:Comparison of total RNA isolated from ASML and ASML CD44v knockdown exosomes; and RNA from untreated B12 lymph node stroma cells vs. cells treated for 24h with ASML wt or ASML CD44v kd exosomes The array consists of 12 samples. Samples 1-3 consist of total RNA from exosomes derived from ASML cells; samples 4-6 : total RNA from exosomes derived from ASML CD44v knockdown cell; samples 7,8 are total RNA from untreated B12 lymph node stroma cells; samples 9,10 are RNA from B12 cells treated for 24h with ASML wt exosomes and samples 11, 12 are RNA from B12 cells treated with ASML CD44v kd exosomes
Project description:Comparison of total RNA isolated from ASML and ASML CD44v knockdown exosomes; and RNA from untreated B12 lymph node stroma cells vs. cells treated for 24h with ASML wt or ASML CD44v kd exosomes
Project description:We hypothesised that eosinophil activation and gene expression were linked to their interaction with the activated stroma within the lymph node. However, the role of eosinophil stromal crosstalk and how this modulate eosinophil function is unknown. To directly assess this hypothesis, we activated lymph node derived stromal cells using an agonist antibody for LTR (Clone 4H8WH2). Considering eosinophils have long been associated with providing B cell proliferative and survival factors, we mimicked a cell-based activation model using purified naïve B cells given that B cell interactions with stroma can produce factors that can regulate eosinophil activation and migration. B cell-based activation as well as agonist antibody-based activation resulted in an activated stromal phenotype and were later used for co-culture with bone marrow-derived eosinophils. After 8 hours of co-culture with activated stroma, the eosinophils were purified, and bulk RNA sequencing was performed to identify global changes in their transcriptomes.
Project description:In this study we focussed our investigations on ECM remodelling by FRCs during lymph node (LN) expansion, and the interconnection between the cellular and ECM components of the conduit network. We demonstrate a loss of ECM components of the conduit during acute LN expansion