Listening to the Whispers in Neuroimmune Crosstalk: A Comprehensive Workflow to Investigate Neurotrophin Receptor p75NTR Under Endogenous, Low Abundance Conditions.

Benjamin W Dorschner,Marc Gentzel,Sebastian Thieme,Mary-Louise Rogers,Ann-Christin Funke,Sebastian Brenner,Ralf Wiedemuth

doi:10.3389/fimmu.2021.648283

Abstract

Inflammatory conditions are critically influenced by neuroimmune crosstalk. Cytokines and neurotrophic factors shape the responses of both nervous and immune systems. Although much progress has been made, most findings to date are based on expression of recombinant (tagged) proteins. The examination of receptor interactions by immunoprecipitation (IP) at endogenous levels provides further insight into the more subtle regulations of immune responses. Here, we present a comprehensive workflow and an optimized IP protocol that provide step-by-step instructions to investigate neurotrophin receptor p75NTR at endogenous, low abundance levels: from lysate preparation and confirmation of receptor expression to antibody validation and successful detection of protein-protein interactions. We employ human melanoma cell line A375 to validate specific antibodies and IP conditions, and apply these methods to explore p75NTR interactions in human leukemic plasmacytoid dendritic cell line PMDC05 detecting 14-3-3ϵ:p75NTR interaction in this cell type. With p75NTR as an exemplary protein, our approach provides a strategy to detect specific interaction partners even under endogenous, low abundance expression conditions.

Highlights

Despite the apparent differences between the nervous and immune systems, they share an intriguing similarity: both employ a system of cytokines and neurotrophic factors enabling them to transmit information from one system to the other [1]
All analyzed exons are expressed in cell lines A375 and PMDC05 with an average expression compared to gapdh of 0.26 and 0.29%, respectively, indicating a low expression in both cell lines
We investigated expression on protein level in melanoma cell line A375 using three different p75NTR specific antibodies, D4B3, ME20.4 and MLR2

Summary

Introduction

Despite the apparent differences between the nervous and immune systems, they share an intriguing similarity: both employ a system of cytokines and neurotrophic factors enabling them to transmit information from one system to the other [1] This neuroimmune crosstalk has been implicated in a wide range of reactions and conditions, e.g. neuroinfectious and autoimmune reactions, as well as allergic. Purified in the late 1970s and early 1980s from rodent sympathetic ganglia and human melanoma cells [9], p75NTR is expressed in central nervous system cells as well as a variety of immune cells [6, 8] Under inflammatory conditions, both NGF secretion and p75NTR expression are dynamically regulated [6, 8]. These dynamic changes shape different immune responses depending on p75NTR expression levels [for review see [10]]

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