Dissecting enteric neuro-immune-microbiome cross-talks using an optogenetic-incorporated gut organ culture system
2Bar-Ilan Institute of Nanotechnology and Advanced Materials, Bar Ilan University, Israel
The enteric nervous system (ENS) neurons and glial cells are embedded within the gut tissue, and are spatially organized in two layers: the myenteric and submucosal plexus, that together, regulates a pleiotropy of gut functions. Recently, the ENS has emerged as an important regulator of immunological responses, inducing microbiome-immune cross-talks; however, the mechanisms underlying enteric neuro-immune communications are mostly enigmatic.
In order to investigate how controlled activation of enteric neurons impact cellular and transcriptional responses of neuronal, as well as non-neuronal post-synaptic cellular targets, we developed a unique optogenetic-incorporated gut organ culture system. This system maintains ex-vivo organ cultures of gut tissues dissected from transgenic mice expressing the light-sensitive ion channel Channelrhodopsin 2 (ChR2) in enteric ChAT+ (Choline acetyl transferase) cholinergic neurons (ChAT+ChR2/YFP+), which are highly abundant in the ENS. Precise activation of enteric cholinergic neurons is achieved by shining blue (activation) light above the tissue (or yellow light, as a control).
Using this system, we have found that short-term optogenetic stimulation by blue light (but not yellow light) induced neuronal activation ex-vivo, as detected by cFos nuclear accumulation in myenteric ChAT+ChR2/YFP+ neurons. Additionally, optogenetic stimulation induced the expression of major cytokines and neuropeptides, including IL-6, IL-17 and VIP (vasoactive intestine peptide), suggesting a role for enteric cholinergic neurons in regulating intestinal immune responses. Overall, we provide a new tool for precise control over neuronal activity in the gut, which is expected to yield novel insights into enteric neuro-immune crosstalk, and their long-term immunological impacts.