The contribution of microbiota in regulating multiple physiological and pathological host responses has been studied intensively in recent years. Evidence suggests that commensal microbiota can directly modulate different populations of cells of the immune system (e.g., Ivanov et al., 2008; Atarashi et al., 2011). Recently, we showed that protein extracts from gut commensal microbiota can activate retina-specific T cells, allowing these autoreactive T cells to then break through the blood-retinal barrier and trigger autoimmune uveitis in the recipient (Horai et al., 2015). The protocol below describes the method to prepare intestinal protein-rich extracts that can be used in various in vitro and in vivo immunological studies.
[Background] Intestinal microbiota represent a complex community of microbes that provide a wide variety of innate and adaptive stimulants. Their isolation and purification from stool samples has been performed and protocols have been published (Mueller and Pan, 2013; Verberkmoes et al., 2009; Tanca et al., 2014; Xiong et al., 2015a; Xiong et al., 2015b). Most of these protocols have been developed with the aim of performing proteomic studies for characterization of the microbiota. Consequently, although they emphasize protein yield and purity, they are time consuming and may include a protein denaturating step that affects protein structure (Verberkmoes et al., 2009) or use reagents (i.e., sodium azide, SDS, phenol) that are incompatible with subsequent cell culture based assays (Tanca et al., 2014; Xiong et al., 2015a; Xiong et al., 2015b). These characteristics are not desired when functional immunological assays are intended to be performed with the extracted proteins.
We have developed a simple and fast method that can be used to obtain protein-rich extracts from different areas of the intestine as well as from stool samples. The protocol does not include denaturating steps and the protein-rich extracts can be used in different in vitro and in vivo immunological assays with live cells, including T cell stimulation for proliferation and for adoptive transfer (see Data analysis section).
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