Tumor necrosis factor alpha derived from classically activated “M1” macrophages reduces interstitial cell of Cajal numbers

• Published in: Neurogastroenterology and motility Vol. 29; no. 4; pp. np - n/a
• Main Authors: Eisenman, S. T., Gibbons, S. J., Verhulst, P. ‐J., Cipriani, G., Saur, D., Farrugia, G.
• Format: Journal Article
• Language: English
• Published: England Wiley Subscription Services, Inc 01.04.2017
• Subjects: Animals; caspase‐mediated apoptosis; Cell Count - methods; cell death; Cells, Cultured; cytokines; diabetic gastroparesis; Dose-Response Relationship, Drug; gastrointestinal motility; Gene Knock-In Techniques; Interstitial Cells of Cajal - drug effects; Interstitial Cells of Cajal - secretion; Macrophages - secretion; Mice; Mice, Inbred BALB C; Tumor Necrosis Factor-alpha - pharmacology; Tumor Necrosis Factor-alpha - secretion; diabetic gastroparesis; gastrointestinal motility; cytokines; caspase-mediated apoptosis; cell death
• ISSN: 1350-1925; 1365-2982
• DOI: 10.1111/nmo.12984

Background Delayed gastric emptying in diabetic mice and humans is associated with changes in macrophage phenotype and loss of interstitial cells of Cajal (ICC) in the gastric muscle layers. In diabetic mice, classically activated M1 macrophages are associated with delayed gastric emptying, whereas alternatively activated M2 macrophages are associated with normal gastric emptying. This study aimed to determine if secreted factors from M1 macrophages could injure mouse ICC in primary culture. Methods Cultures of gastric ICC were treated with conditioned medium (CM) from activated bone marrow‐derived macrophages (BMDMs) and the effect of CM was quantified by counting ICC per high‐powered field. Key Results Bone marrow‐derived macrophages were activated to a M1 or M2 phenotype confirmed by qRT‐PCR. Conditioned medium from M1 macrophages reduced ICC numbers by 41.1%, whereas M2‐CM had no effect as compared to unconditioned, control media. Immunoblot analysis of 40 chemokines/cytokines found 12 that were significantly increased in M1‐CM, including tumor necrosis factor alpha (TNF‐α). ELISA detected 0.697±0.03 ng mL−1 TNF‐α in M1‐CM. Recombinant mouse TNF‐α reduced Kit expression and ICC numbers in a concentration‐dependent manner (EC50 = 0.817 ng mL−1). Blocking M1‐CM TNF‐α with a neutralizing antibody preserved ICC numbers. The caspase inhibitor Z‐VAD.fmk partly preserved ICC numbers (cells/field; 6.63±1.04, 9.82±1.80 w/Z‐VAD.fmk, n=6, P<.05). Conclusions & Inferences This work demonstrates that TNF‐α secreted from M1 macrophages can result in Kit loss and directly injure ICC in vitro partly through caspase‐dependent apoptosis and may play an important role in ICC depletion in diabetic gastroparesis. In diabetic mice, M1 macrophages are associated with delayed gastric emptying, whereas M2 macrophages are associated with normal gastric emptying. TNF‐α, a factor present in M1‐conditioned medium, reduced Kit‐positive ICC numbers and TNF‐α neutralizing antibodies blocked the effect of M1 medium. TNF‐α derived from M1 macrophages injures ICC in vitro and TNF‐α may be important in diseases like diabetic gastroparesis. View the podcast on this paper at the following sites: iTunes: https://itunes.apple.com/gb/podcast/neurogastroenterology-motility-april-2017/id1213419181 Youtube: https://youtu.be/gZfSX890SiE