Unknown,Transcriptomics,Genomics,Proteomics

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RNA-Seq of FACS sorted Enteric Glia cells from the LMMP after T-cell activation via CD3

ABSTRACT: The Experiment was performed to understand the changes in expression profile of enteric glia cells in context of intestinal inflammation. Enteric glia are crucial regulators of gastrointestinal motility, however enteric glia alterations in IBD are poorly characterized. Acute T-cell activation rapidly impacts enteric glia activation, and induces cell death pathways, which negatively impacts small intestinal transit. This experiment has been crucial to identify IFNγ- and TNFα -driven necrosis in activated glia cell subsets. In order to sequence pure enteric glia, a mouse strain of Bl6 mice with a Plp1CreERT insert for glial specificity crossed with mice with a flox controlled tdTomato fluorescent protein on the ROSA26 Locus. The mice received a peritoneal injection of an anti-CD3 antibody or its isotype control antibody. At 6 hours, the small intestine was collected and the longitudinal muscle layer with the myenteric plexus stripped off. After enzymatic dissociation and trituration, the enteric glia were FACS sorted and finally the RNA isolated.

INSTRUMENT(S): Illumina NovaSeq 6000

ORGANISM(S): Mus musculus

SUBMITTER: Miguel Gonzalez Acera

PROVIDER: E-MTAB-14876 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:BACKGROUND Enteric glia contribute to the pathophysiology of various intestinal immune-driven diseases, such as postoperative ileus (POI), a motility disorder and common complication after abdominal surgery. Enteric gliosis of the intestinal muscularis externa (ME) has been identified as part of POI development. However, the glia-restricted responses and activation mechanisms are poorly understood. The sympathetic nervous system becomes rapidly activated by abdominal surgery. It modulates intestinal immunity, innervates all intestinal layers, and directly interfaces with enteric glia. We hypothesized that sympathetic innervation controls enteric glia reactivity in response to surgical trauma. METHODS Sox10iCreERT2/Rpl22HA/+ mice were subjected to a mouse model of laparotomy or intestinal manipulation to induce POI. Histological, protein, and transcriptomic analyses were performed to analyze glia-specific responses. Interactions between the sympathetic nervous system and enteric glia were studied in mice chemically depleted of TH+ sympathetic neurons and glial-restricted Sox10iCreERT2/JellyOPfl/+/Rpl22HA/+ mice, allowing optogenetic stimulation of β-adrenergic downstream signaling and glial-specific transcriptome analyses. A laparotomy model was used to study the effect of sympathetic signaling on enteric glia in the absence of intestinal manipulation. Mechanistic studies included adrenergic receptor expression profiling in vivo and in vitro and adrenergic agonism treatments of primary enteric glial cell cultures to elucidate the role of sympathetic signaling in acute enteric gliosis and POI. RESULTS With ~4000 differentially expressed genes, the most substantial enteric glia response occurs early after intestinal manipulation. During POI, enteric glia switch into a reactive state and continuously shape their microenvironment by releasing inflammatory and migratory factors. Sympathetic denervation reduced the inflammatory response of enteric glia in the early postoperative phase. Optogenetic and pharmacological stimulation of β-adrenergic downstream signaling triggered enteric glia reactivity. Finally, distinct adrenergic agonists revealed β-1/2 adrenoceptors as the molecular targets of sympathetic–driven enteric glial reactivity. CONCLUSIONS Enteric glia act as early responders during post-traumatic intestinal injury and inflammation. Intact sympathetic innervation and active β-adrenergic receptor signaling in enteric glia is a trigger of the immediate glial postoperative inflammatory response. With immune-activating cues originating from the sympathetic nervous system as early as the initial surgical incision, adrenergic signaling in enteric glia presents a promising target for preventing POI development.

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RNA-Seq of FACS sorted Enteric Glia cells from the LMMP after T-cell activation via CD3

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