Intestinal damage is observed in inflammatory bowel disease (IBD), enteric infections,aswellasfollowingsurgicaltrauma,andenvironmentalinsults.Tissuerestitutionisacomplexbut coordinated cellular process to repair gut mucosa. We recently reported that the intestinal wound microenvironment specifically enriches a mucosa-associated microbial consortium, which enhanced epithelial woundhealing.Theadvancesinnextgenerationgenesequencinghaveidentifieddiversemicrobialspeciesand metagenomesinthehost;?however,tounderstandandexploitthepotentialofthemicrobiota?s>8milliongenes andtheirmetabolicfunctions,applicationofthemass-spectrometry-basedidentificationofsmallmetabolitesand functional characterization by the methods of cell biology are crucial. Hence this proposed study will lay a foundationformyrigoroustraininginmetabolomeandcellbiologyofgutbarrierfunction.Forthispurpose,we performedapreliminaryanalysisbymassspectrometry-basedglobalmetabolomicprofilingofintactandinjured mucosa as well as colon of germ-free and conventional mice. We found an elevated synthesis of microbial metaboliteswithlowpKa,whichareessentialinestablishinganextracellularpHgradientinthecoloniccryptsof theconventionalmice,butnotingermfreemice.Wealsofoundthatthecolonicepithelialcellsofconventional miceexpressincreasedlevelsofproton-sensingGPCRs(GPCR4andGPCR68).Basedonpreliminaryfindings, theworkinghypothesesofthisprojectare:1)theinjuredmucosaltissueanditsassociatedmicrobiotaproduce metabolitestoregulatespatiotemporalchangesintheprotonconcentrationofthecolonandtherebyregulate intestinalhomeostasisandrepair,2)microbiota-producedmetabolitesmodulateprotonconcentration,whichis perceivedbytheproton-sensingGPCRsofenterocytestoregulategutpermeability.
Aim1 istoconfirmtherole ofmicrobiota-producedmetabolitesinregulatingcellularproliferation&repairviaproton-sensingGPCR4and GPCR68intheGPCR4-/-andGPCR68-/-mice,whosecoloniesareestablishedinouranimalfacility.Wewillalso perform the metabolomic profiling and RNA-seq analysis of the colon of GPCR4-/- and GPCR68-/- mice. Furthermore,wewilldeterminethemetabolomeoftheinjuredmucosainthegerm-freemouse.
SpecificAim2 willdeterminethefunctionoftheprotongradient&alsotheroleofspecificmetabolitesintheregulationofgut permeability by tight-junction. Emory University boasts an intellectually rich research environment whose resourceswillbeusedtocarryouttheproposedresearchaswellasthemas-spectrometrycenterlocatedacross thetowninGeorgiaInstituteofTechnology,Atlanta.Theproposedresearch,incombinationwithastructured team of mentor, advisors, and collaborators as well as the training plan that includes didactic courses and workshops,isdesignedtofacilitatemylong-termgoalofdevelopinganindependently-fundedresearchprogram inchronicgastrointestinalinflammatorydiseases,suchasIBD,consistentwiththemissionoftheNIDDK.

Public Health Relevance

Importance of extracellular proton concentration is critical in intestinal mucosa during health and disease. However,thereisnoevidence-basedknowledgehowextracellularpHofthemucosalmicroenvironmentregulate intestinalrepairandrestorationofhomeostasis.Leveragingthemetabolomicstudies,thisprojectisdesignedto investigate two proton-sensing G-protein coupled receptors and their role in microbial metabolite-driven extracellularpHgradientduringmucosalhomeostasisandrepair.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Scientist Development Award - Research & Training (K01)
Project #
5K01DK114391-05
Application #
9989830
Study Section
Kidney, Urologic and Hematologic Diseases D Subcommittee (DDK)
Program Officer
Saslowsky, David E
Project Start
2017-08-01
Project End
2021-07-31
Budget Start
2020-08-01
Budget End
2021-07-31
Support Year
5
Fiscal Year
2020
Total Cost
Indirect Cost
Name
University of Kentucky
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
939017877
City
Lexington
State
KY
Country
United States
Zip Code
40526