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538.Full.Pdf The Bacterial Fermentation Product Butyrate Influences Epithelial Signaling via Reactive Oxygen Species-Mediated Changes in Cullin-1 Neddylation This information is current as of September 24, 2021. Amrita Kumar, Huixia Wu, Lauren S. Collier-Hyams, Young-Man Kwon, Jason M. Hanson and Andrew S. Neish J Immunol 2009; 182:538-546; ; doi: 10.4049/jimmunol.182.1.538 http://www.jimmunol.org/content/182/1/538 Downloaded from References This article cites 75 articles, 30 of which you can access for free at: http://www.jimmunol.org/content/182/1/538.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 24, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology The Bacterial Fermentation Product Butyrate Influences Epithelial Signaling via Reactive Oxygen Species-Mediated Changes in Cullin-1 Neddylation1 Amrita Kumar,* Huixia Wu,* Lauren S. Collier-Hyams,* Young-Man Kwon,* Jason M. Hanson,† and Andrew S. Neish2* The human enteric flora plays a significant role in intestinal health and disease. Populations of enteric bacteria can inhibit the NF-␬B pathway by blockade of I␬B-␣ ubiquitination, a process catalyzed by the E3-SCF␤-TrCP ubiquitin ligase. The activity of this ubiquitin ligase is regulated via covalent modification of the Cullin-1 subunit by the ubiquitin-like protein NEDD8. We previously reported that interaction of viable commensal bacteria with mammalian intestinal epithelial cells resulted in a rapid and reversible generation of reactive oxygen species (ROS) that modulated neddylation of Cullin-1 and Downloaded from resulted in suppressive effects on the NF-␬B pathway. Herein, we demonstrate that butyrate and other short chain fatty acids supplemented to model human intestinal epithelia in vitro and human tissue ex vivo results in loss of neddylated Cul-1 and show that physiological concentrations of butyrate modulate the ubiquitination and degradation of a target of the E3- SCF␤-TrCP ubiquitin ligase, the NF-␬B inhibitor I␬B-␣. Mechanistically, we show that physiological concentrations of bu- tyrate induces reactive oxygen species that transiently alters the intracellular redox balance and results in inactivation of the NEDD8-conjugating enzyme Ubc12 in a manner similar to effects mediated by viable bacteria. Because the normal flora http://www.jimmunol.org/ produces significant amounts of butyrate and other short chain fatty acids, these data provide a functional link between a natural product of the intestinal normal flora and important epithelial inflammatory and proliferative signaling pathways. The Journal of Immunology, 2009, 182: 538–546. irtually unique among mammalian cell types, the intes- ucts such as lactate (4, 5). These organic compounds are an im- tinal epithelium coexists in intimate contact with a nor- portant energy source for the colonic epithelium and may influence V mal flora of ϳ1012 prokaryotic organisms. The greatest various aspects of gut physiology. For example, butyrate and other numbers and diversity are in the cecum and ascending colon where SCFAs have well-known differentiating and growth-promoting ac- bacterial density can reach 1011 cells/g of contents, yielding a bio- tivities in vitro and in vivo, a biological effect ascribed to histone by guest on September 24, 2021 mass of Ͼ1 kg. This bacterial community of ϳ500–1000 species deacetylase activity (6). Additionally, SCFAs have been noted to has diverse beneficial roles including vitamin synthesis, bile salt have immunomodulatory effects on colonic inflammation, sup- metabolism, and degradation of complex carbohydrates (1). Fur- pressing inflammatory cytokine secretion in cultured epithelial thermore, through studies with germfree or gnotobiotic mice, it is cells, and ameliorating model colitis in mice, suggesting that these also now established that the enteric flora can fundamentally affect molecules contribute to the ability of the mucosa to tolerate the epithelial gene transcription, ultimately affecting enterocyte pro- presence of vast quantities of living microorganisms and associ- liferation and homeostasis (2, 3). ated microbial-associated molecular patterns (MAMPs) (6, 7). Fur- The normal flora thrives in a largely anaerobic environment, thermore, luminal instillation of butyrate has been shown to be a generating energy by the fermentation of luminal complex carbo- promising experimental therapy in ulcerative colitis and related hydrates. The end products of fermentation are a spectrum of or- inflammatory disorders (8, 9). How these naturally occurring bac- ganic acids, including short chain fatty acids (SCFAs)3 such as terial metabolic products influence epithelial homeostasis are un- butyrate, succinate, and propionate, as well as other terminal prod- known and are a topic of considerable research interest. A key pathway that modulates inflammatory and/or growth sur- vival in the intestine is the NF-␬B or Rel signaling pathway. The *Department of Pathology and Laboratory Medicine, Epithelial Pathobiology Unit, Emory University School of Medicine, Atlanta, GA 30322; and †Department of Pe- NF-␬B pathway is controlled by regulated degradation of a phys- diatrics, Division of Pulmonary, Asthma, Cystic Fibrosis and Sleep, Emory Univer- ically associated inhibitor molecule, I␬B-␣, which when phosphor- sity, School of Medicine, Atlanta, GA 30322 ylated is ubiquitinated by a specific ubiquitin ligase complex des- Received for publication May 27, 2008. Accepted for publication October 9, 2008. ignated E3-SCF␤-TrCP. Ubiquitinated I␬B-␣ is targeted for The costs of publication of this article were defrayed in part by the payment of page degradation by the proteasome (10, 11). E3-SCF␤-TrCP and other charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. E3-SCF complexes are themselves regulated by transient covalent 1 This work was supported in part by National Institutes of Health Grants DK-71604 modifications. The ubiquitin paralog NEDD8 must be conjugated and AI-64462 (to A.S.N.). to the Cullin-1 (Cul-1) subunit of the E3-SCF complex for ubiq- 2 Address correspondence and reprint requests to Dr. Andrew S. Neish, Department uitin ligase activity (12–15). NEDD8 modification of Cul-1 has of Pathology, Emory University School of Medicine, Room 105F, Whitehead Build- ing, 615 Michael Street, Atlanta, GA, 30322. E-mail address: [email protected] 3 Abbreviations used in this paper: SCFA, short chain fatty acid; Cul-1, cullin-1; tiplicity of infection; IAA, iodoacetamide; HMM, high molecular mass; Eh, redox MAMP, microbial-associated molecular pattern; ROS, reactive oxygen species; NAC, potential; Trx, Thioredoxin. N-acetyl cysteine; DPI, diphenyleneiodonium; DCF, 5-(and-6)-chloromethyl-2Ј,7Ј- dichlorodihydrofluorescein diacetate acetyl ester; DHE, dihydroethidium; MOI, mul- Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 www.jimmunol.org The Journal of Immunology 539 been demonstrated to be necessary for ubiquitination of I␬B-␣ and (Sigma-Aldrich) using the enhanced chemiluminescent protocol (ECL; p100/p105, and for the subsequent activation of NF-␬B in mam- Amersham) and a HRP-conjugated secondary Ab. Blots were exposed to malian cells (16–20). filmfor1sto30min. We have demonstrated that viable commensal bacteria can block I␬B-␣ ubiquitination by blockade of neddylation of the Immunofluorescence studies Cul-1 subunit of E3-SCF␤-TrCP, accounting for the attenuation of ␬ Immunofluorescent labeling of p65 in adherent HeLa cells grown on NF- B activation (21). We further reported that loss of Cul-1 ned- 12-mm glass coverslips was performed as follows: cells were fixed for 20 dylation was mediated by rapid and reversible oxidative inactiva- min in 3.7% paraformaldehyde in PBS, washed in PBS, permeabilized with tion of Ubc12, the ubiquitin-like conjugating enzyme responsible 0.1% Triton X-100 in PBS for 5 min, and washed again. Fixed samples for the neddylation of Cullin subunits (22). Herein, we demon- were incubated in blocking solution (5% normal goat serum in PBS) over- strate that butyrate and other SCFAs supplemented to model epi- night at 4°C. A 1-h incubation with each Ab diluted in blocking buffer followed: 1/500 rabbit anti-p65 (Rockland); 1/200 fluorescein (FITC)-con- thelia in vitro and human tissue ex vivo also cause loss of neddy- jugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories). lated Cul-1 resulting in consequent inhibition of the NF-␬B Cells were washed three times between each Ab. The coverslips were pathway. We also show that de novo and transient reactive oxygen mounted on glass slides and stained cells were observed by laser confocal species (ROS) generation mediate these regulatory
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