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I mm un e -m ed i a t e d d i sease and the M ic r o /Ma c r o b i o m e

I mm un e -m ed i a t e d d i sease and the M ic r o /Ma c r o b i o m e. D a v i d E . Elli o t t , M D , P hD. O u t li n e - Lea r n i ng O bject i v es. I m m u n e - m e d i a t ed d i s ea s e a n d t h e E n v i r o n m e n t B r i e f i n t o t o I m m u n e R eg u l a t i on

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I mm un e -m ed i a t e d d i sease and the M ic r o /Ma c r o b i o m e

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  1. Immune-mediateddisease andthe Micro/Macrobiome DavidE.Elliott,MD, PhD

  2. Outline-LearningObjectives • Immune-mediateddiseaseandtheEnvironment • BriefintotoImmuneRegulation • Micro/Macro-biomeasacontextsetter • Microbiomeanddisease • C.difficilecolitisroleofFMT • IBD • Celiac • MS • Helicobacterpylori • Pleaseinterruptandaskquestionsduringtalk • Conversationinstructsbetterthanlecture

  3. Immune-MediatedDisease • Thereareover80well-recognizedimmune-mediateddiseases. Examplesinclude;T1D,RA,MS,Celiac,IBD,Psoriasis,Lupus,Asthma,Grave’s,ITP, … • Most areincreasingin frequencyandarerareinareaswithless industrialization. (fromBach,N.E.J.M.347:911)

  4. InflammatoryBowelDisease NormalColon Crohn’sColitis

  5. Normal Crohn’sDisease DysregulatedInflammation RegulatedInflammation

  6. >160Genes/LociareImplicatedinIBDbyGWAS Both fromLeesGut60:17392011andVanLimbergenNat.Rev.Gastro/Hep11:3722014

  7. EtiologyofIBD Nurture Nature IBD Genes Environment

  8. “Ecologic”EventsinImmunity • GeneticpredispositionexplainsaminorcomponentoftheriskforIBD • ~50%concordanceforCrohn’sdiseasein monozygotictwins • <20%concordanceforUC inmonozygotictwin pairs • (Halfvarson,Gastroenterology124:1767) • Theimmuneresponseissensitivetocontext. ̶ ̶ ̶ ̶ Strength ofreceptorengagement(stochastic selection)Selectionofco-signalingmolecules(perceptionofdanger)Proteoglycanmatrix signals( perceptionoftissuedamage)Pre-existingcytokinemilieu(dictatesresponseprofiles) • TheMicrobiomehelpstosetimmunologiccontext

  9. IL17 ALLERGY AUTOIMMUNITY IL21 TNF IL22 RoRt TNF IL5 IL4 IL13 IL10 IFN IL2 IL6 Th17 Th22 IL6,TGF, IL23,IL1 Th1 Th2 IL12 IL4 APC Th0 TGF IL9 Th9Tr1 TGF nTreg IL10 IL27 Th3 FoxP3 IL2 CD25+ FoxP3 Gata3 IL10 TOLERANCE ANERGY TGF

  10. #Biomics • Current focusisonbacteria • >10,000publicationson gutmicrobiomeinlast5years • ~1000to 1200 bacterialspeciesinhabithuman gut • Each personhas~160 bacterialspeciesin gut(functionalcore) • Healthyidenticaltwinswillhavesimilarbutdifferentbacteriapopulation • Populationisrelatively stablebutcanshift–“PreBiotics” • Dysbiosis=pathologicallyskewed populationofgut bacteria • CausevsEffectofillness • (Lloyd-Price,et.al. ThehealthyhumanmicrobiomeGenomeMed8:512016) • Moreneglectedare theecologicalcontributionof: • Archaea (e.g. Methanobrevibactersmithii) • Virus(+/-RNA,ss/dsDNA,bacteriophages) • Fungi(yeast- Candida,Malassezia,andSaccharomyces) • Protozoa (Blastocystis,Entamoeba,etc...) • Helminths(worldwide50-90%of peoplecarryhelminths) • Oldfriendshypothesis–disappearingbiome

  11. Alteringthe‘BiometoTreatImmune- MediatedInflammatoryDisease(IMID) • Assumptions: • Immunemediateddiseasesare duetoimmunedysregulation • Immunedysregulationis contextual • Themicrobiomehelpssetcontext • Changingthe‘biome maytreatdisease • Examples • C.difficilecolitis(not anIMIDbutbestexampleforbiomeRx) • IBD • Celiac • MS • Helicobacterpylori gastritis

  12. C.difficlecolitis • Plasmid-directedproductionoftoxinsthatmediatedestructionofcolonicepithelium • Causedbyovergrowth ofClostridiumdifficileoftenafterantibiotictreatment • Treatedwithspecificantibioticsbut Clostridiaaresporeformersandrecurrentinfectionsarecommon • Recurrent/refractoryC.difficlecanbetreatedwithFecalMicrobiotaTransplant(FMT) Pseudomembranouscolitis Summitlesion

  13. FMT–refractoryC.diff • AKA-Stooltransplant • FMTis anestablishedtreatmentforRCDI • ~92%effective (betterthanrepeatedvancomycin) • Lowerdelivery ~95%betterthanupperdelivery(~88%) • Nodifferencebetweenfreshorfrozen • Repeatedattemptsaddincrementalefficacy • Donorsneedextensivescreening • (Quraishi,et.al.Systematicreviewwithmeta-analysis:…AlimentPharmacolTher.46:4792017) • OpenBiome(commercialsourceoffrozenpoo) • Youtoocanbecomea stooldonor(Bostononly)!!!! • SynPoop-groups aretrying tocreatestandardizedculturedFM

  14. IBD • IBDdoesnot developingerm-freeanimals • NewonsetpediatricCrohn’sdisease(668patients) • Increasedabundance(Enterobacteriaceae,Pasteurellacaea,Veillonellaceae, andFusobacteriaceae) • Decreasedabundance(Erysipelotrichales,Bacteroidales,andClostridiale) • Dysbiosisisexacerbatedbyantibiotictreatment • (Gevers,et. al.Thetreatment-naïvemicrobiomein new-onsetCrohn’sdisease.CellHostMicrobe.15:382,2014) • Othersfindsimilarchanges(dysbiosis) • CausevsEffectunknown • FMTinIBDisbeingstudied • Repeated(weeklyx6)FMT appearstobeeffectiveinUC (remission:24%FMT 5%Placebo,p=0.03) • Stooldiversityimprovedinrecipients • (Moayyedi,et. al.FecalMicrobiotaTransplantationInducesRemission in PatientsWith ActiveUlcerativeColitis…Gastroenterology149:102,2015) • –NoevidenceyetforefficacyinCrohn’s

  15. RiseinIBD–EasternEurope

  16. EradicationofHookworm • JohnD.Rockefeller initiated • ~1909–SanitaryCommission • Globaleffortassistedby advancesinhygiene • DecreaseinhookworminUSAovertime • Hookwormisno longerendemicinUSA. • Hookwormcouldhelpexplainthepriornorth- south gradient.

  17. HelminthsandtheHumanGenome • Allvertebrateanimalscarryhelminths • Carriageof helminthsappearstohaveinfluencedour genevariation • ITGAV,COL4A1,ITGA8,FLNB,ITGA9,ITGB8,ITGAM,ITGBL1,COL4A2, COL1A2,DOCK2,PTK2B,ITGAX,FYN,MAPK13,LAMA2,ITGAL,LIMS1, COL24A1,ELMO1,COL15A1,DOCK1,MAPK14,COL9A3,VAMP3,SOCS6,PLCH2,PLCB1,GRB2,GNG10,ITPR1,CXCR6,MYH14,ITGA9,MYO3B, PLA2G4B,GNAQ,CAMK2A,PLCL1,CAMK2D,ITGAM,CCR9,COL1A2, ITGB7,PAK7,VAV2,PLCD3,ADCY2,PTK2B,PLCE1,RELA,ITPR2,CCL20, PLA2G4A,COL23A1,IL4,KCNS3,CTLA4,DPP10,TLR4,PTGER2,GATA3, PHF11,IL10,NPSR1,ADRB2,IKZF2,ADRA1A, • Variationincludespathwaysconferringriskforinflammatory and autoimmunedisease (Fumagalli,BMC EvolutionaryBiology10:264,2010)

  18. HelminthsandIBDEpidemiology • Priorhookworminfectionappearstoprotectfrom Crohn’sdisease (Kabeerdoss,Aliment.Pharmacol.Ther.;34:923,2011) • Casecontrol study in Vellore,India:75 Control/ 78 Crohn’s • Assayedin vitro PBMC reactivity tohookwormantigens • IFNELISPOT(+):48%control26%Crohn’sdisease(p<0.005) • • %CD3+CD69+shift:3.16+0.74control,0.90+0.48Crohn’s(p<0.001) • PriorhelminthexposureappearstoprotectfromIBD • (Chu,Inflamm.BowelDis.19:614,2013) • Casecontrol study in CapeTown,SouthAfrica • 88Crohn’sdisease,63ulcerativecolitis,219controls • 56%Crohn’s,66%UC patientsand91%controlsreportedchildhood helminthexposure(p<0.001) • • AdjustedOddsRatioCD=0.2[0.1-0.4], UC=0.2[0.1-0.6]

  19. Heligmosomoides(polygyrus)bakeri • Intestinalnematodeofmice. • completelyentericlifecycle • AcquiredbyingestingsmallL3larvae. • larvaeare about0.3mmlong • Larvaeandadultwormsresideintheduodenum.

  20. BasicH.bakeriprotocol 2 wks Sham Treatment Analyze C57BL/6 2 wks Analyze Hpb L3byGastricLavage

  21. H.bakeriexposurealtersLPMC cytokineexpression ELISAof48hrculturesfromCD3-stimulatedcells.

  22. H.bakeriin CD25-depletiontransfer colitisprotocol Remove CD25+ andB220+cells B6WT Spleen B6reconstitutedRag1-/- B6Rag1-/- Sham 2 wksRx Analyze Piroxicam for2wks 2 wks Analyze HpbL3 Colitic

  23. H.bakeriexposurereverses establishedtransfercolitis 3.78±0.06 p<0.01 1.54±0.13

  24. UlcerativeColitisTrialResults PhaseI PhaseI Response ( UCDAI>4) ChangeinUCDAIComponentScores

  25. CeliacDisease • What is it? • Inappropriateimmuneresponsetogluten WillemKarelDicke • Glutenisatermforstorageproteinsincereal grains • Wheat,Rye,Barley • ?Oats • Delayed-type(TypeIV)hypersensitivityresponse • WheatallergyinitiallydescribedbyWillemDicke • Resultsin chronicintestinalinflammation • Allergyvs. ImmuneMediatedInflammationvs.AutoimmuneDisease

  26. EndoscopicCharacter Celiac Normal

  27. Normal Celiac IEL

  28. Celiacdisease-Genetics MHCClassII&moleculesformdimersthat presentantigenstoTcells. • Morethan95%ofpatientshaveeitherHLA-DQ2(1*5011*0201)ClassIIHLA-DQ8(1*3011*0302)ClassII   • About30%ofthepopulationalsoexpressthesedimers. • DQ2orDQ8arerequiredandcontributebutdonotcauseceliacdisease.

  29. HLA-DQ2bindsglutenpeptides PQPQLPY tTG NH2 PQPELPY P=Proline Q=Glutamine L=Leucine Y=TyrosineE=Glutamate (Kim,PNAS 101:4175,2004)

  30. 5EpitopesinaPoorlyDigestible 33aaGliadinPeptide PQPQLPYPQ PYPQPQLPY ..LQLPFPQPQLPYPQPQLPYPQPQLPYPQPQPF.. P=Proline Q=Glutamine L=Leucine Y=Tyrosine F=Phenylalanine (Shan,Science297:2275,2002)

  31. Celiacandthe‘biome • Casecontrol studyon2,933celiacpatientsmatchedwith28,262controlsfound a positivecorrelation betweenantibiotic use,and subsequentceliac(oddsratio(OR)= 1.40;CI=1.27–1.53). • (Marild,et. al. Antibioticexposureanddevelopment ofcoeliacdiseaseBMC Gastro.13:109,2013) • Duodenalbacteriafromceliacpatientsgiventogerm-freemice alterdigestionofglutentoincreaseimmunopathogenicpeptideavailability. • Pseudomonasaeruginosaseeninceliacincreasedantigenicity • Lactobacillusstrainsfromnon-celiaccontrols reducedantigenicity(Camineroet.al. (2016)Duodenalbacteriafrompatients withceliacdisease…affectgluten breakdownandimmunogenicity.Gastro.151:670,2016) • Celiacpatientshave more G-andfewG+ bacteriaandpopulationschangewithgluten-freediet

  32. Celiacandhelminths • Investigatorsare studyingtheeffectof helminthexposureon celiacdisease. • Utilizedhookworm(Necatoramericanus) • Patientsweregiven20larvae(transdermal)andthenmicro-challenged withlow-dosegluten • Glutenexposuredidnotcauseexpectedduodenalatrophyorincrease anti-tTG,qualityoflifescoresincreased,intestinalTcellIFNγdeclined andFoxp3+Tregsincreased. • “Microbialdiversity”wasenrichedinpatientsharboringN.americanus • (Croeseet. al. ExperimentalhookworminfectionandglutenmicrochallengepromotetoleranceJ AllergyClinImmunol135:508,2015;Giacommet. al.Experimentalhookworminfection…increasedmicrobialrichnessinceliacsubjects.Sci. Reports5:13797,2015)

  33. MultipleSclerosis(MS) • MSis causedbyimmune-mediateddestructionofinsulatingmyelininthecentralnervoussystemcausingaxon damage • Twomajorforms • Relapsing/remittingsecondaryprogressive • Primaryprogressive • Animalmodel– ExperimentalAutoimmuneEncephalitis(EAE) • Microbiome • Nomajorfindingsbutinacasecontrol study;MScasesexhibitedhigherabundancesofMethanobrevibacter(Archaea)andAkkermansiaandlowerButyricimonas • (Jangi,etal.Alterationsof thehuman gutmicrobiomeinMS. Nat.Commun. 7:12015,2016)

  34. MSandhelminths • Casecontrolstudiessuggestthat: • Helminthcarriagereducesdiseaseseverity • (Correale,Associationbetweenparasiteinfectionandimmuneresponses inmultiplesclerosis.Ann Neurol. 61:97.2007) • Eradicationofhelminthsexacerbatesdisease • (Correale,Theimpactofparasiteinfectionson thecourseof multiplesclerosis.J.Neuroimmunol. 233:6,2011) • Therapeutichelminthexposurecanalterimmunecircuits andmayinfluencediseasecourse(open-labelstudy). • (Fleming,etal. Probiotichelminthadministrationinrelapsing-remittingmultiplesclerosis: a phase1study. MultScler.17:743,2011)

  35. HelicobacterpyloriGastritis/Duodenitis • MotileGram(-)spiral-shapedrodbacteria • Mostcommoninfectionworldwide(>50%) • Infectionassociatedwithdecreasedsocioeconomicstatus • Prevalencevariesbyageandgroup(nextslide) • Mostinfectionsareasymptomatic

  36. Helicobacterpylori

  37. H.pylori andPUD • 2005 • DiscoveredasagentofPUD • Marshall&Warren.Lancet8390:13111984 • H.pyloricausesbothacuteandchronicgastritis • Most(~90%)ofpatientswithPUDandnotusingNSAIDshaveH.pylori. • Lifetimeriskvaries3%(US)to25%(Japan) • Hp+ NSAIDsincreasesPUDrisk60fold • EradicationofH.pylorireducesulcerrecurrence • – from~90%to10%.

  38. H.pylori andpepticulcer disease • H.pyloridoesnotinvade(remainslumenal) • H.pylorisecretesenzymesandtoxins • AllmakeUrease • Splitsureainto bicarbonate+ NH3 • Buffersacid • MoststrainsmakeVacAandCagA • VacAcausesvacuolationofepithelialcells • CagAincreasescytokineproductionbyepithelialcells • Uncertaincontributiontopathogenicity • Immuneresponseiswhatcauses disease • –ChronicinflammationdrivesPUDandotherpathology

  39. H.pylori –OtherIllness • AtrophicGastritis • CommoncauseofB12deficiencyintheelderly • GastricCancer(Adenocarcinoma) • H.pyloriisclassifiedasType1(definite)carcinogen • Themajorcauseofgastriccancerworldwide • MALTLymphoma(98%haveH.pylori) • MALT = Mucosa-associatedLymphoidTissue • B-celltumorinfiltratingthegastricmucosa • About80%regresswithH.pylorieradication

  40. H.pylori diagnosis/treatment • Diagnosis • Serology(IgGAnti-Hpantibody) • Best screeningtestif on PPI • Remains+ for~2yrs afterRx • Ureasebreathtest • StoolAgtest • Gastricbiopsy(evaluationofPUD) Giemsa,KacarInternetJournalofPathology.3:2004 • Histology • urease“CLO”testing • Treatment • Iffound–> treat. • Eradicationrateisabout75% • Triple therapy(PPI+2antibioticsfor2weeks) • ClarithromycinplusAmpicillinorMetronidazole

  41. HelicobacterpyloriMicrobiome • Eradication hasnoeffectongeneraldiversitybut • reducesrelativeabundanceofBacterioidetes • withcorrespondingincreaseinFirmicutes • (Wan-ChenYap,etal.Helicobacter pyloriEradicationCausesPerturbationof the HumanGutMicrobiomeinYoungAdults. PlosOne11:e0151893,2016) • Otherbacteriainhabitthestomach • H.pyloripathologymaybe influencedbyotherbugs • (ShehTheroleof thegastrointestinalmicrobiomein Helicobacterpyloripathogenesis.GutMicrobes.4:505,2013) • HelminthinfectionaltersH.pylori pathologyandmay decreasecancerrisk • (Ek, et al. Serologicevidencethatascarisandtoxoplasmainfectionsimpactinflammatoryresponses toHelicobacterpyloriinColombians.Helicobacter.17:107-15,2012)

  42. Immune-mediateddisease andthe Micro/Macrobiome Questions?

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