Background: Alpha7 nicotinic acetylcholine receptor (α7nAChR) is mainly expressed in central nervous system to play important roles, such as memory formation. However, in recent years, accumulating evidences show that α7nAChR is also expressed in immune cells, such as macrophages, and activation of α7nAChR induces anti-inflammatory action . We previously found myenteric neuron-mediated anti-inflammatory action via α7nAChR in small intestine using postoperative ileus model (Gut, 60; 638) . However, target cells expressed α7nAChR in small intestine are not well identified, because there is not a good antibody for the receptor.
Aim: Here we identify α7nAChR-expressing target cells in healthy and inflamed condition in small intestine.
Materials and Methods: We generated α7nAChR-FLAG-tag mice by using CRISPR/Cas9 system. We performed magnetic cell sorting to isolate macrophages subset. Postoperative ileus model was used as an intestinal inflammation.
Results: In healthy condition, α7nAChR mRNA was expressed more highly in intestinal muscle layer than mucosal layer. CD11b+Gr-1- macrophages subset in intestinal muscle layer expressed α7nAChR mRNA, but not in mucosal layer. In immunohistochemical analysis of α7nAChR-reporter mice, FLAG-tag+CD68+ macrophages could be detected in muscle layer, suggesting that intestinal muscularis resident macrophages expressed α7nAChR. On the other hand, FLAG-tag+ cells in mucosal layer were CD68-, suggesting that α7nAChR-expressed cells in mucosal layer is not macrophages. However, in inflamed condition of postoperative ileus, CD11b+Gr-1- macrophages subsets in mucosal layer expressed α7nAChR mRNA. Moreover, immunohistochemical analysis revealed that FLAG-tag+CD68+ macrophages could be detected in both muscle and mucosal layer. Circulating monocytes did not express α7nAChR mRNA in both healthy condition.
Conclusion: Intestinal muscularis, but not mucosal resident macrophages express α7nAChR. In the inflamed condition, mucosal macrophages also express α7nAChR. Although circulating monocytes do not express α7nAChR, infiltrated monocyte-derived macrophages express the receptors. Based on these, it is suggested that macrophages subsets expressed α7nAChR dramatically changed during inflammation.