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Background Postoperative ileus (POI) is a common complication of abdominal surgery and is marked by impaired gut motility driven by inflammation of the muscularis externa. While muscularis macrophages (mMfs) are central to this response, the early trigger(s) responsible for their activation remain unclear.Objective To assess the role of the mechanosensitive neurons and enteric glial cells (EGCs) in the early activation of mMfs in POI.Design Intestinal manipulation (IM) was performed in wild-type C57BL/6 mice and the kinetics of cell influx and activation were assessed. Single-nucleus RNA sequencing (snRNA-seq) was conducted on the intestinal muscularis layer 30 min after sham and IM. In vitro and in vivo studies were conducted to validate the role of Ccl4 and Cxcl10 in mMf activation. D-GsMTx4 was administered while Wnt1Cre; Piezo2fl/wt mice were generated to analyse the role of Piezo2 in POI.Results Inflammatory cells, primarily mature mMfs, increased in the muscularis externa within 30 min post-IM due to cell proliferation, while cFos staining showed enteric neurons and EGCs to be activated prior to mMfs. Immunostaining revealed expression of Piezo2 in the enteric neurons and EGCs and inhibition of Piezo2 significantly reduced IM-induced inflammation. snRNAseq indicated cell-cell communication between mMfs and mMfs/EGCs via the release of Ccl4 and Cxcl10, respectively. Finally, blockade of Ccl4 and Cxcl10 attenuated mMf activation.Conclusions Our findings reveal activation of Piezo2-expressing enteric neurons and EGCs as a very early step in IM-induced inflammation of POI. These cells subsequently activate mMfs resulting in upregulation of Ccl4, further amplifying the inflammatory cascade. These results highlight a key role for Piezo2 in the pathophysiology of POI.