Background Intestinal barrier failure may lead to systemic inflammation and distant organ injury in patients following severe injury. Enteric glia cells (EGCs) have been shown to play an important role in maintaining gut barrier integrity through secretion of S-Nitrosoglutathione (GSNO). We have recently shown than Vagal Nerve Stimulation (VNS) increases EGC activation, which was associated with improved gut barrier integrity. Thus, we sought to further study the mechanism by which EGCs prevent intestinal barrier breakdown utilizing an in vitro model. We postulated that EGCs, through the secretion of GSNO, would improve intestinal barrier function through improved expression and localization of intestinal tight junction proteins. Methods Epithelial cells were co-cultured with EGCs or incubated with GSNO and exposed to Cytomix (TNF-α, INF-γ, IL-1β) for 24 hours. Barrier function was assessed by permeability to 4kDa FITC-Dextran. Changes in tight junction proteins ZO-1, occludin, and phospho-MLC (P-MLC) were assessed by immunohistochemistry and immunoblot. Key Results Co-culture of Cytomix-stimulated epithelial monolayers with EGCs prevented increases in permeability and improved expression and localization of occludin, ZO-1, and P-MLC. Further, treatment of epithelial monolayers with GSNO also prevented Cytomix-induced increases in permeability and exhibited a similar improvement in expression and localization of occludin, ZO-1, and P-MLC. Conclusions & Inferences The addition of EGCs, or their secreted mediator GSNO, prevents epithelial barrier failure after injury and improved expression of tight junction proteins. Thus, therapies that increase EGC activation, such as VNS, may be a novel strategy to limit barrier failure in patients following severe injury.
References
[1]
Musch MW, Walsh-Reitz MM, Chang EB (2006) Roles of ZO-1, occludin, and actin in oxidant-induced barrier disruption. Am J Physiol Gastrointest Liver Physiol 290: G222-G231. doi:10.1152/ajpgi.00301.2005. PubMed: 16239402.
[2]
Madara JL, Moore R, Carlson S (1987) Alteration of intestinal tight junction structure and permeability by cytoskeletal contraction. Am J Physiol 253: C854-C861. PubMed: 3425707.
[3]
Shen L, Black ED, Witkowski ED, Lencer WI, Guerriero V et al. (2006) Myosin light chain phosphorylation regulates barrier function by remodeling tight junction structure. J Cell Sci 119: 2095-2106. doi:10.1242/jcs.02915. PubMed: 16638813.
[4]
Deitch EA, Shi HP, Lu Q, Feketeova E, Skurnick J et al. (2004) Mesenteric lymph from burned rats induces endothelial cell injury and activates neutrophils. Crit Care Med 32: 533-538. doi:10.1097/01.CCM.0000109773.00644.F4. PubMed: 14758175.
[5]
Krzyzaniak MJ, Peterson CY, Cheadle G, Loomis W, Wolf P et al. (2011) Efferent vagal nerve stimulation attenuates acute lung injury following burn: The importance of the gut-lung axis. Surgery 150: 379-389. doi:10.1016/j.surg.2011.06.008. PubMed: 21783215.
[6]
Costantini TW, Bansal V, Peterson CY, Loomis WH, Putnam JG et al. (2010) Efferent vagal nerve stimulation attenuates gut barrier injury after burn: modulation of intestinal occludin expression. J Trauma 68: 1349-1354. doi:10.1097/TA.0b013e3181dccea0. PubMed: 20539179.
[7]
Costantini TW, Loomis WH, Putnam JG, Drusinsky D, Deree J et al. (2009) Burn-induced gut barrier injury is attenuated by phosphodiesterase inhibition: effects on tight junction structural proteins. Shock 31: 416-422. doi:10.1097/SHK.0b013e3181863080. PubMed: 18791495.
[8]
Costantini TW, Loomis WH, Putnam JG, Kroll L, Eliceiri BP et al. (2009) Pentoxifylline modulates intestinal tight junction signaling after burn injury: effects on myosin light chain kinase. J Trauma 66: 17-24. doi:10.1097/TA.0b013e3181937925. PubMed: 19131801.
[9]
Costantini TW, Peterson CY, Kroll L, Loomis WH, Putnam JG et al. (2009) Burns, inflammation, and intestinal injury: protective effects of an anti-inflammatory resuscitation strategy. J Trauma 67: 1162-1168. doi:10.1097/TA.0b013e3181ba3577. PubMed: 20009662.
[10]
Samonte VA, Goto M, Ravindranath TM, Fazal N, Holloway VM et al. (2004) Exacerbation of intestinal permeability in rats after a two-hit injury: burn and Enterococcus faecalis infection. Crit Care Med 32: 2267-2273. PubMed: 15640640.
Shiou SR, Yu Y, Chen S, Ciancio MJ, Petrof EO et al. (2011) Erythropoietin protects intestinal epithelial barrier function and lowers the incidence of experimental neonatal necrotizing enterocolitis. J Biol Chem 286: 12123-12132. doi:10.1074/jbc.M110.154625. PubMed: 21262973.
[13]
Bansal V, Costantini T, Ryu SY, Peterson C, Loomis W et al. (2010) Stimulating the central nervous system to prevent intestinal dysfunction after traumatic brain injury. J Trauma 68: 1059-1064. doi:10.1097/TA.0b013e3181d87373. PubMed: 20453760.
[14]
Krzyzaniak M, Peterson C, Loomis W, Hageny AM, Wolf P et al. (2011) Postinjury vagal nerve stimulation protects against intestinal epithelial barrier breakdown. J Trauma 70: 1168-1176. doi:10.1097/TA.0b013e318216f754. PubMed: 21610431.
[15]
Costantini TW, Bansal V, Krzyzaniak M, Putnam JG, Peterson CY et al. (2010) Vagal nerve stimulation protects against burn-induced intestinal injury through activation of enteric glia cells. Am J Physiol Gastrointest Liver Physiol 299: G1308-G1318. doi:10.1152/ajpgi.00156.2010. PubMed: 20705905.
[16]
Rühl A, Nasser Y, Sharkey KA (2004) Enteric glia. Neurogastroenterol Motil 16 Suppl 1 Suppl 1: 44-49. doi:10.1111/j.1743-3150.2004.00474.x. . PubMed : 15066004.
[17]
Savidge TC, Sofroniew MV, Neunlist M (2007) Starring roles for astroglia in barrier pathologies of gut and brain. Lab Invest 87: 731-736. doi:10.1038/labinvest.3700600. PubMed: 17607301.
[18]
Neunlist M, Van Landeghem L, Bourreille A, Savidge T (2008) Neuro-glial crosstalk in inflammatory bowel disease. J Intern Med 263: 577-583. doi:10.1111/j.1365-2796.2008.01963.x. PubMed: 18479256.
[19]
Savidge TC, Newman P, Pothoulakis C, Ruhl A, Neunlist M et al. (2007) Enteric glia regulate intestinal barrier function and inflammation via release of S-nitrosoglutathione. Gastroenterology 132: 1344-1358. doi:10.1053/j.gastro.2007.01.051. PubMed: 17408650.
[20]
Bansal V, Costantini T, Kroll L, Peterson C, Loomis W et al. (2009) Traumatic brain injury and intestinal dysfunction: uncovering the neuro-enteric axis. J Neurotrauma 26: 1353-1359. doi:10.1089/neu.2008.0858. PubMed: 19344293.
[21]
Wang Q, Guo XL, Wells-Byrum D, Noel G, Pritts TA et al. (2008) Cytokine-induced epithelial permeability changes are regulated by the activation of the p38 mitogen-activated protein kinase pathway in cultured Caco-2 cells. Shock 29: 531-537. PubMed: 17724435.
[22]
Chavez AM, Menconi MJ, Hodin RA, Fink MP (1999) Cytokine-induced intestinal epithelial hyperpermeability: role of nitric oxide. Crit Care Med 27: 2246-2251. doi:10.1097/00003246-199910000-00030. PubMed: 10548215.
[23]
Boivin MA, Ye D, Kennedy JC, Al-Sadi R, Shepela C et al. (2007) Mechanism of glucocorticoid regulation of the intestinal tight junction barrier. Am J Physiol Gastrointest Liver Physiol 292: G590-G598. PubMed: 17068119.
[24]
Costantini TW, Deree J, Loomis W, Putnam JG, Choi S et al. (2009) Phosphodiesterase inhibition attenuates alterations to the tight junction proteins occludin and ZO-1 in immunostimulated Caco-2 intestinal monolayers. Life Sci 84: 18-22. doi:10.1016/j.lfs.2008.10.007. PubMed: 18992758.
[25]
Neunlist M, Aubert P, Bonnaud S, Van Landeghem L, Coron E et al. (2007) Enteric glia inhibit intestinal epithelial cell proliferation partly through a TGF-beta1-dependent pathway. Am J Physiol Gastrointest Liver Physiol 292: G231-G241. PubMed: 16423922.
[26]
Conlin VS, Wu X, Nguyen C, Dai C, Vallance BA et al. (2009) Vasoactive intestinal peptide ameliorates intestinal barrier disruption associated with Citrobacter rodentium-induced colitis. Am J Physiol Gastrointest Liver Physiol 297: G735-G750. doi:10.1152/ajpgi.90551.2008. PubMed: 19661153.
[27]
Neunlist M, Toumi F, Oreschkova T, Denis M, Leborgne J et al. (2003) Human ENS regulates the intestinal epithelial barrier permeability and a tight junction-associated protein ZO-1 via VIPergic pathways. Am J Physiol Gastrointest Liver Physiol 285: G1028-G1036. PubMed: 12881224.
[28]
Costantini TW, Krzyzaniak M, Cheadle GA, Putnam JG, Hageny AM et al. (2012) Targeting α-7 nicotinic acetylcholine receptor in the enteric nervous system: A cholinergic agonist prevents gut barrier failure after severe burn injury. Am J Pathol 181: 478-486. doi:10.1016/j.ajpath.2012.04.005. PubMed: 22688057.
[29]
MacEachern SJ, Patel BA, McKay DM, Sharkey KA (2011) Nitric oxide regulation of colonic epithelial ion transport: a novel role for enteric glia in the myenteric plexus. J Physiol 589: 3333-3348. doi:10.1113/jphysiol.2011.207902. PubMed: 21558161.
[30]
Borovikova LV, Ivanova S, Zhang M, Yang H, Botchkina GI et al. (2000) Vagus nerve stimulation attenuates the systemic inflammatory response to endotoxin. Nature 405: 458-462. doi:10.1038/35013070. PubMed: 10839541.
Tracey KJ (2007) Physiology and immunology of the cholinergic antiinflammatory pathway. J Clin Invest 117: 289-296. doi:10.1172/JCI30555. PubMed: 17273548.
[33]
Rosas-Ballina M, Olofsson PS, Ochani M, Valdés-Ferrer SI, Levine YA et al. (2011) Acetylcholine-synthesizing T cells relay neural signals in a vagus nerve circuit. Science 334: 98-101. doi:10.1126/science.1209985. PubMed: 21921156.
[34]
Van Landeghem L, Chevalier J, Mahé MM, Wedel T, Urvil P et al. (2011) Enteric glia promote intestinal mucosal healing via activation of focal adhesion kinase and release of proEGF. Am J Physiol Gastrointest Liver Physiol 300: G976-G987. doi:10.1152/ajpgi.00427.2010. PubMed: 21350188.
[35]
Van Landeghem L, Mahé MM, Teusan R, Léger J, Guisle I et al. (2009) Regulation of intestinal epithelial cells transcriptome by enteric glial cells: impact on intestinal epithelial barrier functions. BMC Genomics 10: 507. doi:10.1186/1471-2164-10-507. PubMed: 19883504.
[36]
Flamant M, Aubert P, Rolli-Derkinderen M, Bourreille A, Neunlist MR et al. (2011) Enteric glia protect against Shigella flexneri invasion in intestinal epithelial cells: a role for S-nitrosoglutathione. Gut 60: 473-484. doi:10.1136/gut.2010.229237. PubMed: 21139062.
[37]
Jaffrey SR, Erdjument-Bromage H, Ferris CD, Tempst P, Snyder SH (2001) Protein S-nitrosylation: a physiological signal for neuronal nitric oxide. Nat Cell Biol 3: 193-197. doi:10.1038/35055104. PubMed: 11175752.
[38]
Marshall HE, Hess DT, Stamler JS (2004) S-nitrosylation: physiological regulation of NF-kappaB. Proc Natl Acad Sci U S A 101: 8841-8842. doi:10.1073/pnas.0403034101. PubMed: 15187230.
[39]
Reynaert NL, Ckless K, Korn SH, Vos N, Guala AS et al. (2004) Nitric oxide represses inhibitory kappaB kinase through S-nitrosylation. Proc Natl Acad Sci U S A 101: 8945-8950. doi:10.1073/pnas.0400588101. PubMed: 15184672.
[40]
Al-Sadi R, Ye D, Dokladny K, Ma TY (2008) Mechanism of IL-1beta-induced increase in intestinal epithelial tight junction permeability. J Immunol 180: 5653-5661. PubMed: 18390750.
