Team:Stanford/Project Homeostasis/Inflammation Device

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Project
Research Proposal
Systems Overview
Cloning Plan
Sequences & Primers
Anti-Inflammation
Device Overview
Parts Design
Challenges
Results
Anti-Immunosuppression
Device Overview
Parts Design
Challenges
Results
Protocols
Modeling
Overview
Notebook
Results
Future Work
Archived Work



Contents

Summary of Device

File:Device 1 Overview.jpg


The anti-inflammatory device consists of two components: a sensor and a responder. The sensor portion detects an input molecule whose concentrations reflect the degree of local inflammation. When the concentration of the input rises above a certain baseline threshold, the sensor will activate the responder portion and induce the production of an output molecule. The output will attenuate local inflammation by enhancing the expansion of the immunosuppressive Treg lineage while curbing the proliferation of the inflammatory Th17 lineage.

We have chosen the soxRS regulon, a genetic defense mechanism native to E. coli, to serve as our sensor. The soxRS regulon is responsive to nitric oxide (NO), a free radical that exists at high concentrations in the inflamed lumen of patients with active inflammatory bowel diseases. In our machine, the presence of NO activates the constitutively expressed soxR protein, forming a complex that binds to the soxS promoter and induces production of the output molecule, all-trans retinoic acid (RA). Synthesis of RA requires the Crt cluster and the blh gene, which together constitute a carotenoid biosynthetic pathway that metabolizes isoprenoid precursors and yields RA. RA diffuses across the E. coli membranes and negatively regulates the Th17 lineage while enhancing the Treg response. Thus, in response to high concentrations of the input signal NO, the soxRS sensor will activate the anti-inflammatory responder and induce production of RA.

Parts Designs

Page Covering Our Parts Designs


Other Considerations

NO toxicity to E. coli: As a free radical, NO is toxic to cells and has been shown to disrupt cellular respiration of E. coli at high concentrations. However, we do not anticipate this to be a problem given the sensitivity of the protective mechanisms that have evolved in E. coli to protect against growth inhibition by NO. In particular, the enzymes cytochrome bd and flavohemoglobin have been implicated in this protection and are sensitive to NO at micromolar concentrations.

RA toxicity to host: High concentrations of vitamin A are toxic and chronic overdose (hypervitaminosis A) is associated with pathologic liver conditions and osteoporosis. However, the recommended daily allowance for vitamin A is 900 micrograms, an amount well above the threshold needed to polarize T cell lineages. Acute toxicity is associated with an intake of > 100x the RDA in the course of a few days for adults and chronic toxicity correlates with a daily intake of preformed vitamin A 7500 micrograms for six years.

Other potential "input" molecules: PGE2

Other possible “output” molecules: vitamin D, Y-320 pyrazoleanilide derivative, 17beta-oestradiol


Challenges


Results


Sources

RA regulation of Th17/Tregs

  • Elias et al. Retinoic acid inhibits Th17 polarization and enhances FoxP3 expression through a Stat-3/Stat-5 independent signaling pathway. Blood. 2008 Feb 1;111(3):1013-20.
  • Mucida et al. Reciprocal TH17 and regulatory T cell differentiation mediated by retinoic acid. Science. 2007 Jul 13; 317(5835):256-60.
  • Xiao et al. Retinoic acid increases Foxp3+ regulatory T cells and inhibits development of Th17 cells by enhancing TGF-beta-driven Smad3 signaling and inhibiting IL-6 and IL-23 receptor expression. J Immunol. 2008 Aug 15;181(4):2277-84.

RA toxicity and therapy

  • Bai et al. All-trans retinoic acid down-regulates inflammatory responses by shifting the Treg/Th17 profile in human ulcerative and murine colitis. J Leukoc Biol. 2009 May 28.
  • Mora. Homing imprinting and immunomodulation in the gut: role of dendritic cells and retinoids. Inflamm Bowel Dis. 2008 Feb;14(2):275-89.
  • [http://ods.od.nih.gov/factsheets/vitamina.asp#h3 Penniston et al.] The acute and chronic toxic effects of vitamin A. Am J Clin Nutr. 2006 Feb;83(2):191-201.
  • [http://ods.od.nih.gov/factsheets/vitamina.asp#h3 NIH Office of Dietary Supplements] Dietary Supplement Fact Sheet: Vitamin A and Carotenoids.

RA synthesis

  • [http://www.jbc.org/cgi/content/full/284/23/15781 Kim et al.] In vitro characterization of a recombinant Blh protein from an uncultured marine bacterium as a β-carotene 15,15′-dioxygenase. J Biol Chem. 2009 Jun 5;284(23):15781-93. (Look at Supplement for precise sequence.)

Resistance to NO in E. coli

  • [http://www.jbc.org/cgi/content/full/M002471200#B25 Stevanin et al.] Flavohemoglobin Hmp affords inducible protection for Escherichia coli respiration, catalyzed by cytochromes bo' or bd, from nitric oxide. J Biol Chem. 2000 Nov 17; 275(46): 35868-75.
  • [http://www.nature.com/nchembio/journal/v5/n2/abs/nchembio.135.html Mason et al.] Cytochrome bd confers nitric oxide resistance to Escherichia coli. Nature Chemical Biology 5, 94 - 96 (2008)
  • [http://www.sciencedirect.com.laneproxy.stanford.edu/science?_ob=ArticleURL&_udi=B6T36-3PNRX2B-53&_user=145269&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000012078&_version=1&_urlVersion=0&_userid=145269&md5=2dfcadd4335175d43afd4be999bbb49e Yu et al.] Oxygen-dependent regulation of the respiration and growth of Escherichia coli by nitric oxide. FEBS Lett. 1997 Jun 9;409(2):161-5.
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