Pulmonary neuroimmune determinants of immunoglobulin production against pneumonia - Project Summary. Community acquired pneumonia cases are predominantly caused by infections from Streptococcus pneumoniae. Evidence shows that effective adaptive immunity in the form of adequate antibody production is necessary to protect against pulmonary S. pneumoniae infection. Emerging evidence demonstrates that the sensory nervous system regulates innate and cellular immunity. Our data and others show that sensory neurons are necessary for antibody production. Yet, the neuronal involvement in adaptive and humoral immunity is under-investigated. Therefore, there is an urgent need to understand the neural regulation of humoral immunity. This application aims to investigate the role of sensory neurons in B cell maturation, immunoglobulin production including class switch recombination, and interaction with neutrophils in response to pulmonary S. pneumoniae infection. Our preliminary data have led us to hypothesize that in response to pulmonary S. pneumoniae infection, sensory neurons release select neuropeptides to activate B cell immunoglobulin release, to interact with neutrophils to clear infection. Secondly, we hypothesize that this downstream effect results from sensory neurons being able to detect specific antigens. We show that 1) sensory neuron neuropeptide release is directly involved in maturing and stimulating B cells in the lungs; 2) Sensory neuropeptides stimulate immunoglobulin production in vitro and in vivo; 3) Sensory neurons appear to assist with B cell-neutrophil-interaction to assist with bacterial clearance. With regards to neuronal antigen sensing, we show that 1) Sensory neurons harbor RNA required for antigen sensing; 2) Antigen receptor proteins are upregulated in sensory neurons with repeat S. pneumoniae exposure; 3) Sensory neuron stimulation with antigen increases neuronal excitation and select neuropeptide release which has immunogenic properties and; 4) Neurons sequester immunoglobulins from B cells. We will investigate this novel neuronal circuit of B cell immunoglobulin production and neutrophil interaction with two aims. Studies in AIM 1 will investigate B cell development and maturation using flow cytometry, class-switch recombination utilizing flow cytometry and tandem single-cell RNAseq and V(D)J repertoire analysis, and neutrophil interaction using in vivo and in vitro assays, including single-cell RNAseq and flow cytometry. Studies in AIM 2 will use electrophysiological recording, calcium imaging, and flow cytometry to assess a novel sensory neuron antigen recognition signaling pathway and what this stimulation means regarding neuropeptide release. Subsequent molecular assays will delineate the exact downstream effector molecule to identify potential drug targets for future study. Finally, targeted antigen receptor deletion in sensory neurons with genetic models will establish the necessity of neuronal antigen sensing to contribute to humoral immunity. Disease severity in all aims will be assessed by enumeration of bacterial burden and immunoglobulin analysis. This contribution is significant because it is expected to elucidate the role of sensory neurons in pulmonary adaptive immunity.
