Tuberculosis alters immune-metabolic pathways resulting in ...Dec 17, 2020  · M.tb infection and...

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1 Tuberculosis alters immune-metabolic pathways resulting in perturbed IL-1 responses Alba Llibre 1 , Nikaïa Smith 1 , Vincent Rouilly 2 , Munyaradzi Musvosvi 3 , Elisa Nemes 3 , Céline Posseme 1,4 , Simba Mabwe 3 , Bruno Charbit 5 , Stanley Kimbung Mbandi 3 , Elizabeth Filander 3 , Hadn Africa 3 , Violaine Saint-André 1,6 , Vincent Bondet 1 , Pierre Bost 4,7 , Humphrey Mulenga 3 , Nicole Bilek 3 , Matthew L Albert 8 , Thomas J Scriba 3 , Darragh Duffy* 1,4. 1 Translational Immunology Lab, Institut Pasteur, Paris, France 2 DATACTIX, Paris, France 3 South African Tuberculosis Vaccine Initiative (SATVI), Division of Immunology, Department of Pathology and Institute of Infectious Disease and Molecular Medicine, University of Cape Town, South Africa 4 Sorbonne Université, Complexité du vivant, F-75005 Paris, France 5 Cytometry and Biomarkers UTechS, CRT, Institut Pasteur, Paris, France 6 Bioinformatics and Biostatistics HUB, Computational Biology Department, Institut Pasteur, USR 3756 CNRS, Paris, France 7 Systems Biology Group, Center for Bioinformatics, Biostatistics, and Integrative Biology (C3BI) and USR 3756, Institut Pasteur CNRS, 8 Insitro, San Francisco, California, USA. * Corresponding author Darragh Duffy Translational Immunology Lab Institut Pasteur 25, rue du Dr. Roux 75724 Paris Cedex 15 France Tel: +33 1 44 38 93 34 Fax: + 33 1 45 68 85 48 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this this version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.17.423082 doi: bioRxiv preprint

Transcript of Tuberculosis alters immune-metabolic pathways resulting in ...Dec 17, 2020  · M.tb infection and...

  • 1

    Tuberculosisaltersimmune-metabolicpathwaysresultinginperturbed

    IL-1responses

    AlbaLlibre1,NikaïaSmith1,VincentRouilly2,MunyaradziMusvosvi3,ElisaNemes3,Céline Posseme1,4, Simba Mabwe3, Bruno Charbit5, Stanley Kimbung Mbandi3,ElizabethFilander3,HadnAfrica3,ViolaineSaint-André1,6,VincentBondet1,PierreBost4,7,HumphreyMulenga3,NicoleBilek3,MatthewLAlbert8,ThomasJScriba3,DarraghDuffy*1,4.1TranslationalImmunologyLab,InstitutPasteur,Paris,France2DATACTIX,Paris,France3SouthAfricanTuberculosisVaccineInitiative(SATVI),DivisionofImmunology,DepartmentofPathologyandInstituteofInfectiousDiseaseandMolecularMedicine,UniversityofCapeTown,SouthAfrica4SorbonneUniversité,Complexitéduvivant,F-75005Paris,France5CytometryandBiomarkersUTechS,CRT,InstitutPasteur,Paris,France6BioinformaticsandBiostatisticsHUB,ComputationalBiologyDepartment,InstitutPasteur,USR3756CNRS,Paris,France7SystemsBiologyGroup,CenterforBioinformatics,Biostatistics,andIntegrativeBiology(C3BI)andUSR3756,InstitutPasteurCNRS,8Insitro,SanFrancisco,California,USA.*Correspondingauthor

    DarraghDuffy TranslationalImmunologyLab InstitutPasteur 25,rueduDr.Roux 75724ParisCedex15 France Tel:+33144389334 Fax:+33145688548

    preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.17.423082doi: bioRxiv preprint

    https://doi.org/10.1101/2020.12.17.423082

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    SUMMARY

    Tuberculosis (TB) remains a major public health problem with host-directed

    therapeutics offering potential as novel treatment strategies. However, their

    successful development still requires a comprehensive understanding of how

    Mycobacterium tuberculosis (M.tb) infection impacts immune responses. To

    address this challenge, we applied standardised immunomonitoring tools to

    compare TB antigen, BCG and IL-1β induced immune responses between

    individualswithlatentM.tb infection(LTBI)andactiveTBdisease,atdiagnosis

    andaftercure.ThisrevealeddistinctresponsesbetweenTBandLTBIgroupsat

    transcriptomic, proteomic and metabolomic levels. At baseline, we identified

    pregnanesteroidsandthePPARγpathwayasnewimmune-metabolicdriversof

    elevated plasma IL-1ra in TB. We also observed dysregulated induced IL-1

    responses after BCG stimulation in TB patients. Elevated IL-1 antagonist

    responseswereexplainedbyupstreamdifferencesinTNFresponses,whileforIL-

    1agonistsitwasduetodownstreamdifferencesingranzymemediatedcleavage.

    Finally, the immune response to IL-1β driven signallingwas also dramatically

    perturbedinTBdiseasebutwascompletelyrestoredaftersuccessfulantibiotic

    treatment.Thissystemsimmunologyapproachimprovesourknowledgeofhow

    immune responses are altered during TB disease, and may support design of

    improveddiagnostic,prophylacticandtherapeutictools.

    Keywords:Tuberculosis,Interleukin1(IL-1),Interleukin1Receptorantagonist(IL-1ra),Immunometabolism.

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    INTRODUCTION

    As the leading cause of death by infection, tuberculosis (TB) is amajor global

    publichealthproblem1.Itisestimatedthatonefourthoftheworld’spopulationis

    infectedbyitscausativeagent,Mycobacteriumtuberculosis(M.tb).M.tbinfection

    results in a diverse clinical spectrum, which includes asymptomatic latent

    infection(LTBI),incipientorsubclinicalstages,andactiveTBdisease(TB),which

    occursin5-10%ofinfectedpersons2,3.

    M.tbaltersthehost’sabilitytoclearinfectionbytargetingimmuneandmetabolic

    pathways. Initial research in mice, later supported by human studies4,

    demonstrated that interferon γ (IFNγ), tumour necrosis factor (TNF) and

    interleukin (IL)-1β are essential cytokines for immune control of M.tb5–8.

    Signalling through the IL-1 receptor can be regulated by binding of the IL-1

    receptor antagonist (IL-1ra)9. Levels of circulating IL-1ra are elevated in TB

    patients,andhavebeenproposedasapotentialbiomarkerfordiagnosisofactive

    diseaseorresponsetotreatment10.However,littleisknownabouttheunderlying

    mechanisms that lead to higher IL-1ra concentrations in active TB disease.

    Metabolic reprogramming is also crucial for determining successful immune

    responses11. Furthermore, immune-metabolic signatures associated with TB

    progressionhavepreviouslybeendescribed12,13.

    Tobetterunderstandhowthesecrucialcytokinepathwaysareperturbedinactive

    TBdisease,we applied a standardised immunomonitoring tool14,15 to compare

    induced immune responses between LTBI and TB patients at proteomic,

    transcriptomic,andmetabolomiclevels.Relevantstimulithatwereusedincluded

    M.tbantigens(TBAg),BacillusCalmette-Guérin(BCG),IL-1b,andaNullcontrol.

    Thisapproachrevealedmultipledifferencesattheproteomicandtranscriptomic

    levelsbetweenLTBIandTBpatients.Integrationofinducedcytokineresponses

    withbaselinemetabolicprofileshelpedtoidentifyuniqueimmuneandmetabolic

    driversofelevatedplasmaIL-1rainTBpatients,inparticular,thePPARγpathway.

    AfterBCGstimulation,IL-1rawassecretedathigherlevelsinactiveTBpatients

    compared to LTBI. Experimental analysis revealed that thiswas partly due to

    differences in TNF mediated signalling. Furthermore, IL-1α and IL-1β were

    secreted at lower levels in active TB patients due to differences in post-

    translationalmodifications.Finally,IL-1βstimulationrevealedadysregulatedIL-

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    1 signalling response in TB patients, which was restored after successful

    treatment.Overall,ourapproachaddsnewunderstandingofhowM.tb impacts

    human immune responses, providing new avenues for better diagnostic tools,

    vaccinesandtreatments,includinghost-directedtherapeutics.

    RESULTS

    Immune stimulation identifies specific gene expression differences that

    improvestratificationofactiveTBdiseasefromlatentM.tbinfection

    ToexaminehowTBdiseaseperturbs immune responses,we stimulatedwhole

    blood from active TB patients and LTBI controls with relevant stimuli that

    includedTBAg,BCG and IL-1β, plus a non-stimulated control (Null). Principal

    componentanalysis(PCA)of622immunegenesmeasuredshowedclusteringof

    stimuli-specific responses,with54%of the total variance capturedby the first

    three principal components (Figure 1A and Suppl Figure 1A). As previously

    reported16, several genes were differentially expressed in active TB disease

    compared to LTBI in the absence of stimulation, namely 32% (200 genes, q

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    high number of differences between LTBI andTBpatients,we testedwhether

    standardised immune stimulation could improve the performance of these

    signatures by comparing ROC curve analysis from Null and TB Ag stimulated

    samples(Figure1Dand1E;SupplFigure1Dand1E).FortheSweeney3signature,

    TB Ag stimulation accentuated the pre-existing differences at baseline, which

    translatedintoasuperiorareaunderthereceiveroperatingcharacteristiccurve

    (AUC)forNullAUCof0.82[95%CI(0.66-0.94)]andforTBAgAUCof0.95[95%CI

    (0.88-1)]improvingtheabilitytoclassifyLTBIandTBcases(p=0.02)(Figure1E).

    Similar results were obtained with the RISK11 signature, with NullAUC of 0.82

    [95% CI (0.69-0.94)] and TB AgAUC of 0.97 [95% CI (0.93-1)] (p=0.02) (Suppl

    Figure 1E). These differences were not present after successful antibiotic

    treatmentoftheTBpatientsintheSweeney3signature(Figure1F;NullAUC=0.63

    [95%CI (0.45-0.82)] andTBAgAUC=0.51 [95%CI (0.32-0.71)], (p=0.4)or the

    RISK11signature(SupplFigure1F;NullAUC=0.57[95%CI(0.38-0.77)]andTB

    AgAUC = 0.63 [95% CI (0.45-0.81)] (p=0.7), confirming the specificity of these

    signatures.

    In summary, immunestimulation revealedmultipledifferences between latent

    M.tbinfectionandactiveTBdiseasethatlikelyreflectrelativedifferencesinISG

    expressioninbloodleukocytes,whichmayhelptobetterunderstandperturbed

    immunity during TB pathogenesis and potentially improve new diagnostic

    strategies.

    TheIL-1pathwayisdysregulatedinactiveTBdisease

    The TB Ag stimulation illustrated in Figure 1D and 1E captures differences in

    antigen-specific responses of CD4+ T cells, and subsequent downstream

    responses,betweenLTBIandTBpatients.ToexplorethebroaderimpactofTB

    disease in mounting efficient immune responses to complex stimuli, we

    investigatedtheresponsetoBCG. Toidentifyimmunepathwaydifferences,we

    performed correlation analysis between BCG-induced cytokines at the protein

    level (FC>1.3) in the LTBI and TB groups (Figure 2A). This revealed negative

    correlationsinthecontrolLTBIgroup,thatwereabsentormarkedlyweakerin

    TBpatients,suggestingalteredregulationofcytokineproductioninTBdisease.

    ThisincludedseveralkeycytokinesimportantforimmuneresponsesagainstTB

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    suchas IL-1α, IL-1β, IL-10,GM-CSF (CSF2)andTNF thatall showedapositive

    correlationwiththeIL-1receptorantagonist(IL-1ra) inTBpatients,butnot in

    LTBI(Figure2A).

    Because IL-1 signalling is essential for M.tb control8,19, we further explored

    differenceswithintheIL-1familymembersbetweenLTBIandTB.Westartedby

    investigating levels of IL-1ra and observed that TB patients presented higher

    concentrationsofIL-1rabothatbaselineandafterBCGstimulation(Figure2B).

    To better understand the drivers of IL-1ra secretion at baseline in TB, we

    performed correlation analysis of the protein data comprising 40

    cytokines/chemokinesintheNulltubeandshowedthatthestrongestassociation

    (r=0.52, p=0.0001) was with IL-8 (Figure 2C), a cytokine which also had

    significantlyhigherlevelsinTBpatients(Figure2D).ExpressionlevelsofbothIL-

    8 receptors (CXCR1 and CXCR2)were also higher in TB patients (Figure 2E),

    howeverstimulationofbloodfromhealthycontrolswithIL-8didnotinduceIL-

    1ra (Figure 2F). We next explored gene expression signatures commonly

    associated with these two cytokines and found genes related to the

    peroxisomeproliferator-activatedreceptor (PPAR)γ pathway (Figure 2G). We

    validated our findings using a published dataset of transcriptional analysis of

    human monocyte-derived macrophages (hMDMs) infected with different

    bacterial strains20. This showed a weak, yet significant positive correlation

    (r=0.34,q=0.03)betweenIL1RN(thetranscriptforIL-1ra)andPPARGtranscripts

    inhMDMsinfectedwithpathogenicmycobacteria,butnotwithnon-mycobacterial

    species(r=-0.28,q=0.1)(Figure2H).Theseresultsidentifyanimmuno-metabolic

    linkbetweenthePPARγpathwayandthereceptorantagonistIL-1ra.

    PregnanesteroidsactivatethePPARγpathwayresultinginincreasedIL-1ra

    secretioninactiveTB

    PPARγ is a ligand-activated transcription factor that plays an essential role in

    metabolismandenergyhomeostasis,particularlyinadipogenesis21,22.Toexplore

    potential metabolic pathways involved in IL-1ra secretion, we performed

    unsupervised mass spectrometry analysis on supernatants from unstimulated

    LTBIandTBpatients’bloodandexaminedwhichmetabolitescorrelatedwithIL-

    1ra protein at baseline in the TB group. Interestingly, 5 of the top 6 ranked

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    metabolites showing significant correlations (r>0.6, q≤0.05)with IL-1ra in TB

    patientsbelongedtopregnanesteroidderivatives(Figure3AandSupplementary

    Table5).ThesecorrelationswereobservedinTBpatients,andnotinLTBI(Figure

    3Aand3B).Interestingly,levelsofthesesteroidsdidnotdifferbetweentheLTBI

    andTBgroups(SupplFig2A),incontrasttoPPARGexpression,whichwashigher

    inTB(SupplFig2B).Pregnanesteroidshavebeenshowntobepotentialligands

    ofPPARγ23, andPPARγ ismainlyexpressedbyCD14+monocytes (HumanCell

    Atlas).ToinvestigatewhetheractivationofPPARγcoulddriveIL-1rasecretion,

    weincubatedCD14+monocytesisolatedfromhealthydonors,withcombinations

    of the PPARγ agonist rosiglitazone and the PPARγ antagonist GW9662, in the

    presenceofBCG.Wevalidatedourapproachbymeasuringsurfaceexpressionof

    CD36, a fatty acid translocase that is up-regulated by PPARγ24–26. CD14+

    monocytes stimulated with rosiglitazone upregulated CD36, and this could be

    prevented with GW9662 (Suppl Fig 2C). We observed that PPARγ activation

    through rosiglitazone increased IL-1ra secretion by CD14+ monocytes, which

    couldbeinhibitedbyGW9662(Figure3C).TofurtherconfirmtheroleofPPARγ

    inIL-1rasecretion,wesilencedPPARγusingsmallinterferingRNA(siRNA).We

    confirmedPPARγknockdowninCD14+monocytesbymeasuringlevelsofPPARγ

    protein by flow cytometry (Suppl Fig 2D). Both at baseline and after BCG

    stimulation,silencingofPPARγresultedindecreasedIL-1rasecretion(Figure3D),

    withthisknockdownimpactingbothconditionsequally(Figure3E).Thissuggests

    that the PPARγ pathway is not involved specifically in BCG-induced IL-1ra,

    although these data confirm PPARγ as a key regulator of baseline IL-1ra

    productioninTBdisease.

    IncreasedTNFsignalingpromotesIL-1rasecretion

    ProteinlevelsofIL-1rawerehigherinTBpatientsbothatbaselineandafterBCG

    stimulation(Figure2B),thelatterofwhichwasnotexplainedbyPPARγ(Figure

    3E).Therefore,we investigatedthedriversofIL-1rasecretion inthecontextof

    BCGstimulation.PreviousstudieshaveidentifiedTNF,IL-1andtypeIandIIIFNs

    asdriversofIL-1rasecretion27–32.Totestthisinoursystemwestimulatedwhole

    bloodfromhealthy individualswithTNF, IL-1β, IFNγ, IFNβ, IFNα,andIL-8and

    confirmedTNFand IFNβ (q=0.001)as the strongest inducersof IL-1ra (Figure

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    4A).Furthermore,TNFandtypeIIFNsactedsynergisticallyininducingIL-1ra,as

    the combinationof these cytokines (TNF+ IFNα/β) resulted ina4-foldhigher

    inductionascomparedtostimulationwitheachcytokinealone(Figure4B).We

    theninvestigatedtheeffectofblockingIFNandTNFsignallingthroughtheuseof

    blockingantibodies.BlockingofTNFR(q=0.001),butnotIFNAR(q=0.7),resulted

    insignificantlylessIL-1raproduction(Figure4C).Toobtainfurtherinsightinto

    thepotentialcontributionofTNFandtypeIIFNinIL-1raproductionuponBCG

    stimulation,weexaminedapreviouslypublishedkineticstudyofBCGstimulated

    wholeblood33.Thisconsistedoffivehealthydonorsat13timepointswithin30h

    of BCG stimulation from which we measured IFNα and IFNβ to analyse with

    existingTNFandIL-1radata.Atime-dependentlinearregressiononIL-1rausing

    IFNα,IFNβandTNFproteinsecretionaspredictorsrevealedastrongassociation

    withTNF(q=0.002)butnottypeIIFNs(IFNαq=0.6,IFNβq=0.6)(Figure4D).IFNβ

    wasnotinduceduponBCGstimulation,andIFNαwasinducedbyBCGfromthe

    10htimepoint(Figure4D).

    To apply these findings to TB patients, we measured TNF protein after BCG

    stimulation and observed that levels of TNFwere surprisingly higher in LTBI

    compared to TB (Figure 4E). We next examined expression levels of the TNF

    receptorsubunit(TNFRSF1B)andobservedsignificantlyhigherlevels(q=0.0001)

    in TBpatients (Figure 4F),whichmay reflect increased signalling. To test this

    hypothesis,wecalculatedagenescorebasedonuniquecytokine-inducedgene

    signatures(seeMaterialsandMethodsandSupplementaryTable6)aspreviously

    described15.TheTNFgenescore,whichisasurrogateforquantifyingsignallingby

    thiscytokine,washigherintheTBgroupcomparedtoLTBIafterBCGstimulation

    (Figure4G),andalsopositivelycorrelated(Rs=0.32,p=0.03)withIL-1raprotein

    concentrations(Figure4H).TheseresultsdemonstrateanessentialroleforTNF

    inBCG-inducedIL-1ra,aresponsethatisaugmentedinactiveTBdisease.

    TBpatientspresentlowergranzymeconcentrationswhichimpactlevelsof

    functionalIL-1αandIL-1β

    BCGstimulationrevealeddysregulationofmultiplearmsoftheIL-1pathwayin

    activeTBdisease(Figure2A).AswellasobservingdifferentiallyinducedIL-1ra

    betweenLTBIandTB(Figure2B),wealsosawsignificantdifferencesinIL-1αand

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    IL-1βsecretion(Figure5A). Incontrast to IL-1ra,bothIL-1αandIL-1βprotein

    were secreted at significantly higher levels in LTBI upon BCG stimulation.

    However,thesedifferenceswerenotmirroredatthetranscriptlevel(Figure5B).

    We therefore hypothesized that post-transcriptional/post-translational

    modificationsmightbe impairedduringactiveTBdisease. IL-1βrequirespost-

    translationalcleavagetosignalandengage functionaloutcomes,whereas IL-1α

    canmanifest basal levels of activity in its unprocessed form34. As this protein

    processing canoccur through either inflammasome-dependent or independent

    manners34,35,we exploredwhether both pathwayswere engaged in ourwhole

    bloodstimulationsystem.WestimulatedbloodfromhealthydonorswithBCGin

    combinationwitheitheraninflammasomeinhibitor(theSykinhibitorR406)ora

    serine-protease inhibitor (3,4-Dichloroisocoumarin, DCI) capable of blocking a

    range of enzymes involved in inflammasome-independent cleavage of IL-1

    immatureproteins,includinggranzymesandcathepsins.Bothinhibitorsresulted

    in a partial but significant reduction of IL-1β secretion after BCG stimulation

    (Figure5C),supportingarolefortheinflammasomeandserine-proteasesinthe

    secretionofIL-1βinstimulatedwholebloodcultures.

    WethenexamineddifferencesinthesepathwaysbetweenLTBIandTBpatients.

    The transcripts of inflammasome components NLRP3, SYK, CARD9 and CASP1

    weresignificantlymoreexpressedafterBCGstimulationinTBpatientscompared

    toLTBI(Figure5D).CathepsinG,aneutrophilserine-proteasethatcleavespro-IL-

    1β,wasalsoexpressedathigherlevelsintheTBgroup(Figure5E),whereasno

    differenceswereobservedincathepsinCtranscripts.Incontrast,othergenesthat

    participateininflammasome-independentIL-1cleavage,suchasgranzymesA,B

    andK,wereexpressedathigherlevelsintheLTBIgroup(Figure5F)suggesting

    that thispathwaywasmore relevant forBCGinduced IL-1β.Concentrationsof

    granzymeBintheBCGsupernatantswerehigherintheLTBIgroupcomparedto

    TB,however,nosignificantdifferenceswereobservedwithgranzymeA(Figure

    5G).ItwasnotablethatmostgranzymeAlevelswerebelowthelimitofdetection

    of the assay. Levels of granzyme B strongly correlated with IL-1α (R=0.53,

    p=0.0001)andtherewasamoderatebutsignificant(R=0.33,p=0.02)correlation

    between IL-1β protein and Granzyme A (Figure 5H). These data support our

    hypothesisofaberrantIL-1inductioninTBdisease,withlowerlevelsofgranzyme

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    proteinresultinginreducedcleavageofIL-1precursors,andthuslessactiveforms

    ofIL-1cytokines.

    IL-1βsignallingisperturbedinactiveTBdiseaseandisresetaftersuccessful

    antibiotictreatment

    WhileBCGstimulationrevealedperturbedsecretionofIL-1cytokinesinactiveTB

    disease(Figure2Band5A),itdidnotallowexaminationofpotentialdifferences

    inIL-1signalling.Toinvestigatethis,westimulatedwholebloodfromTBpatients

    andLTBIcontrolswithIL-1βanddefinedasignatureof107inducedgenes(Null

    vsIL-1βq

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    forM.tbcontrol,areperturbedinTBdisease.Thepotentialclinicalrelevanceof

    such an approachwas illustrated by the observation that immune stimulation

    improved the diagnostic score of a previously described gene expression

    signatureforTBdiagnosis17.

    TheantagonistIL-1ramoleculewaselevatedinTBatbaselineconditions.Despite

    thiscytokinehavingbeenproposedasapotentialbiomarkertodistinguishLTBI

    from active TB10, the biological triggers of IL-1ra in the context of TB remain

    largelyunknown.WeshowedthatincreasedbasallevelsofIL-1rainTBpatients

    are associated with the PPARγ pathway. PPARγ is constitutively and highly

    expressedinhealthyalveolarmacrophages39,40,andhasbeenshowntoregulate

    hostimmuneandmetabolicresponsestomycobacterialinfection41.M.tb-driven

    activationofPPARγpromotesIL-8secretion42,andactivationofPPARγinTHP-1

    cells results in IL-1ra secretion43, in agreement with our observations.

    Furthermore, Pott and colleagues identified IL1RN as a putative PPARγ target

    geneinaTHP-1modelofhumanmacrophages44.Weproposeamodelinwhich

    pregnanesteroids inTBdiseaseactivatethePPARγpathway,whichmayactat

    differentlevels(eg.CD36,IL-1ra)topromoteM.tbreplication.M.tbcanpromote

    CD36 expression through PPARγ activation45, and CD36 contributes to M.tb

    survival46,47.Finally,thePPARγaxiscantriggerIL-1rasecretion43,whichinhibits

    IL-1signalling,essentialforpathogenclearance8,19.InhibitionofPPARγhasbeen

    associated with decreased mycobacterial burden in mouse and human

    macrophages41,42,45,anddeletionofPPARγinlungmacrophageshasbeenshown

    to be immunoprotective in the context of M.tb infection in mice48. PPARγ is

    therefore a good host-directed therapeutic target candidate, as it impacts lipid

    bodybiogenesis,cytokineproductionandM.tbreplication49.PPARγagonistsare

    alreadybeingusedinthetreatmentofconditionssuchasdiabetes,wheretheyact

    as insulin sensitizers50. Diabetes prevalence is escalating globally and the

    increasing overlapwithTB is amajor source of concern, with type 2 diabetic

    patients having a 3-fold increased risk of developing TB51. Activating PPARγ

    agentswould be predicted to enhance susceptibility to TB disease. Therefore,

    careful assessment of how metabolic modulators impact comorbidities is

    required.

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    StimulationwithBCGrevealedperturbed IL-1 family cytokine responses inTB

    patients,which iscrucial forhostcontrolofM.tb infection7,52,53.Specifically,TB

    patients simultaneously had lower agonist (IL-1a and IL-1β) and higher

    antagonist (IL-1ra) responses. Detailed kinetic studies33, combinedwith single

    cytokinestimulations15,blockingexperiments,andtimeseriesanalysispermitted

    theidentificationofTNFaskeydriverofIL-1rainTBdisease.Theseresultsaddto

    the existing complexities of cytokine responses toM.tb, in which the overall

    contextwilldetermine infectionoutcome, rather than thepresence/absenceof

    particularcytokines.Forinstance,thetimingofIL-154,55ortypeIIFNsignalling56

    isadecisivefactortoexertbeneficialrolesinthecontextofTBdisease.Ourstudy

    shows how TNF, a critical cytokine for control of M.tb, also induced IL-1ra

    expression,whichcanpreventeffectiveIL-1responses,thatareinturnessential

    forprotectiveimmunity.

    The observed IL-1a and IL-1β protein differences were not mirrored at the

    transcriptional level, suggesting defects in either post-transcriptional or post-

    translationalregulation.Notably,bothIL-1aandIL-1βrequireenzymaticpost-

    translational cleavage,which canoccur through inflammasome-dependentor–

    independent mechanisms57. M.tb has previously been shown to inhibit

    inflammasome activation and IL-1β processing58, and a recent study

    demonstrated that CARD9, an inflammasome protein associated with fungal

    induction of proinflammatory cytokines, negatively regulated IL-1βproduction

    duringbacterialinfection59.IthasalsobeenshownthatIL-1βproductioninM.tb

    infection can take place independently of caspase-1 and ASC-containing

    inflammasomesinmice53.Inourstudyweobservedbothhigherinflammasome

    activity and lower granzyme expression in TBpatients. Thus, lower granzyme

    activityinTBpatientsmayresultinlowerpost-translationalcleavageofIL-1a/β.

    This is supported by previous studies in which PBMC stimulation withM.tb-

    related antigens resulted in lower levels of granzyme B in TB patients60. In

    addition, a recent multi-cohort study identified granzyme B, produced by

    polyfunctional NK cells, as a characterising feature of LTBI compared to

    uninfectedcontrols61.

    WeandothershavereporteddistinctsecretionofIL-1agonistsandantagonists

    uponM.tbinfection8,10,62,however,whetherIL-1signallingitselfisimpairedinTB

    preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.17.423082doi: bioRxiv preprint

    https://doi.org/10.1101/2020.12.17.423082

  • 13

    diseaseremainspoorlyexplored.Here,weshowedperturbedIL-1βsignallingin

    TBpatients,withhigherexpressionofmostIL-1β-inducedgenesinTBcompared

    toLTBI.ThisobservationshowsthatthereisnointrinsicdefectinIL-1βsignalling

    capabilitiesinTBpatients.HoweverthetimingofIL-1responseshasbeenshown

    tobecriticalforM.tbcontrol54,55,andelevatedIL-1signallinghasbeenassociated

    with lung immunopathology63,64. The consequences of IL-1β hyper

    responsiveness in TB patients therefore warrants further investigation.

    Importantly,theelevatedIL-1βsignallingwasnolongerpresentaftersuccessful

    antibiotic treatment, suggesting that immune responseswere reset to healthy

    levels.Alimitationofthecurrentstudyisthatexperimentswereperformedon

    peripheralblood,whereasthecytokinenetworksthatregulateM.tb infectionin

    vivoalsoactontissueresidentcells,whichmightbehavedifferently.Also,cellular

    differencesbetweenthegroupsstudied,suchashighermonocyte-to-lymphocyte

    and neutrophil-to-lymphocyte ratios in active TB compared to LTBI65, could

    potentiallycontributetosomeoftheobserveddifferences.

    In summary, our study of immune-metabolic responses revealed perturbed

    responses in TB disease at transcriptomic, proteomic andmetabolomic levels.

    Integration of these datasetshas improved our knowledge on howTBdisease

    impacts immune-metabolic pathways resulting in perturbed IL-1 responses,

    whichareessentialforM.tbcontrol.ThisbetterunderstandingofhowM.tbalters

    immune responses may facilitate the improved design of preventive and

    therapeutictools,includinghost-directedstrategies.

    ACKNOWLEDGEMENTS

    ThisstudywasfundedbytheBillandMelindaGatesFoundation(OPP1114368

    and OPP1204624), with additional support from the French Government’s

    Investissement d’Avenir Program, Laboratoire d’Excellence “Milieu Intérieur”

    Grant ANR-10-LABX-69-01. AL was supported by the Fondation Recherche

    Médicale (SPF20170938617) and the European Commision (H2020-MSCA-IF-

    2018, 841729). NS was supported by an Institut Pasteur Roux Cantarini

    fellowship.We thank the UTechS CB of the Center for Translational Research,

    InstitutPasteurforsupportingNanostringanalysis.DDthanksImmunoqurefor

    preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.17.423082doi: bioRxiv preprint

    https://doi.org/10.1101/2020.12.17.423082

  • 14

    provisionofthemAbsunderanMTAfortheSimoaIFN-αassay.Wearegratefulto

    thestudyparticipantsandtheSATVIclinicalandlaboratoryteams.

    AUTHORCONTRIBUTIONS

    AL designed and performed experiments, analysed and interpreted data and

    wrotethemanuscript.NSdesignedandperformedexperiments,andanalysedand

    interpreteddata.VRanalyseddata.MMandENperformedexperimentsontheTB

    patientcohort.MM,CP,SM,BC,SK,VSA,VB,PB,HMandNBperformedspecific

    experimentsand/oranalysis.EFandHAweretheclinicaland laboratorystudy

    coordinatorsinSouthAfrica,respectively.MLA,TJSandDDconceivedthewhole

    study, obtained funding and provided overall guidance. DD designed and

    supervisedthewholestudy,designedexperiments,analysedandinterpreteddata

    andwrotethemanuscript.Allauthorscontributedtomanuscriptrevision,read

    andapprovedthesubmittedversion.

    DECLARATIONOFINTERESTS

    MLAisacurrentemployeeofInsitro,whohadnoinfluenceonthestudydesignor

    reporting.Theotherauthorsdeclarenocompetinginterests.

    FIGURELEGENDS

    Figure1.Immunestimulationidentifiesspecificgeneexpressiondifferences

    betweenlatentM.tbinfectionandactiveTBdisease.(A)Principalcomponent

    analysis(PCA)onexpressionof622genesfrom24individualswithlatentM.tb

    infection(LTBI)and24withactiveTBdisease(TB)afterwholebloodstimulation

    withTBAg,BCG,IL-1β,andanon-stimulatedcontrol(Null).Eachcolouredcircle

    represents one individual for each condition. (B) Upset plot showing the

    intersection of differentially expressed genes (q value

  • 15

    ofLTBI/TBgroupsusingSweeney3fortheNullandTBAgconditionsonvisit1

    pre-treatment (Pre-Tx) (E) and on visit 2 after successful antibiotic treatment

    (Post-Tx)(F).ComparisonsofLTBI/TBgroupswithinthesamestimulationwere

    performedusingunpairednon-parametrict-tests;comparisonsbetweenNulland

    stimulatedconditionswithintheLTBI/TBgroupswereperformedusingapaired

    non-parametrict-test.Correctionformultiplecomparisonswasthenapplied.Red

    line:medianvalues.Closedsquare:LTBI,Opentriangle:TB.

    SupplementaryFigure1.(A)Screeplotshowingthevarianceexplainedbyeach

    PCA componenton thegene expression (Nanostring) dataset. (B) Examples of

    significantlydifferent(q

  • 16

    proteinconcentrationmeasuredbyELISA insupernatantsofwholebloodfrom

    healthyindividualsstimulatedwithIL-8.(G)Venndiagramofthegenescorrelated

    with IL-1ra and IL-8 in theNull condition inTB,measured byNanostring and

    Luminex,respectively.GenesassociatedwiththePPARγpathwayareinbold.(H)

    ReanalysisofRNAseqdatabySpearmancorrelationbetweenPPARGandIL1RN

    geneexpression levelsofhumanmonocyte-derivedmacrophages infectedwith

    pathogenic mycobacteria and non-mycobacteria species for 18 and 48h20.

    Comparisonsbetweengroupswereperformedusingunpairedorpaired(F)non-

    parametrict-testsandcorrectionformultiplecomparisonswasapplied.Redline:

    median values. Closed square: LTBI, Open triangle: TB, Closed circle: Healthy

    Control(HC).

    Figure 3. Pregnane steroids activate the PPARγ pathway resulting in

    increasedIL-1rasecretion inactiveTB. (A)HeatmapofPearsoncorrelation

    coefficients between metabolites and IL-1ra protein in the Null condition,

    measuredbymassspectrometryandLuminex,respectively,forTB(left)andLTBI

    (right). Metabolites are ordered in decreasing values of TB cases Pearson

    correlationcoefficients.(B)PearsoncorrelationbetweenIL-1ralevelsmeasured

    by Luminex and 5α-pregnane-3β,20α-diol disulfate measured by mass

    spectrometry for LTBI and TB. (C) IL-1ra protein concentration measured by

    ELISAafterstimulationofCD14+monocytesfromhealthyindividualswithBCGin

    the presence or absence of the PPARγ agonist rosiglitazone (Rosi) and/or the

    PPARγantagonistGW9662.(D)IL-1raproteinconcentrationmeasuredbyELISA

    afterstimulationwithBCGofPPARGsilencedorcontrolCD14+monocytesfrom

    healthyindividuals.(E)RatioofIL-1rainduceduponBCGstimulationversusthe

    Null Control in PPARG silenced or control CD14+ monocytes. Comparisons

    between groups were performed using paired non-parametric t-tests and

    correctionformultiplecomparisonswasapplied.Redline:medianvalues.Closed

    square:LTBI,Opentriangle:TB,Circle:HealthyControl(HC).

    SupplementaryFigure2.(A)Relativelevelsofpregnanesteroidsidentifiedin

    Figure 3A for both LTBI and TB, measured by mass spectrometry in the Null

    condition.(B)GeneexpressionlevelsofPPARGintheNullconditionmeasuredby

    preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.17.423082doi: bioRxiv preprint

    https://doi.org/10.1101/2020.12.17.423082

  • 17

    Nanostring both in LTBI andTB. (C) Protein levels of CD36measured by flow

    cytometry on the surface of CD14+monocytes fromhealthy individuals in the

    presenceorabsenceofthePPARγagonistrosiglitazone(Rosi)and/orthePPARγ

    antagonistGW9662.(D)IntracellularproteinlevelsofPPARγmeasuredbyflow

    cytometry in CD14+monocytes from healthy individuals after treatment with

    PPARGsmall interferingRNA(siRNA)orcontrol.Comparisonsbetweengroups

    wereperformedusingunpaired(A,B)orpaired(C)non-parametrict-testsand

    correctionformultiplecomparisonswasapplied.Redline:medianvalues.Closed

    square:LTBI,Opentriangle:TB,Circle:HealthyControl(HC).

    Figure 4. Increased TNF signaling promotes IL-1ra secretion. (A)

    Concentrationsof IL-1rameasuredbyLuminexafterstimulationofbloodfrom

    healthyindividualswithTNF,IL-1β,IFNγ,IFNα,IFNβ,andIL-8.(B)IL-1raprotein

    concentrationmeasuredbyELISAafterwholebloodstimulationwithTNF,IFNα,

    IFNβ,TNF+IFNαandTNF+IFNβ.(C)IL-1raproteinconcentrationmeasuredby

    ELISAafterwholebloodstimulationwithBCG in thepresenceof anti-TNFRor

    anti-IFNAR,ortheirrespectiveisotypecontrols.(D)KineticsofIL-1ra,IFNα,IFNβ

    andTNFsecretionmeasuredbyLuminex(IL-1raandTNF)andSimoa(IFNαand

    IFNβ)uponBCGstimulationduringthecourseof30h.1representativedonorof

    five healthy individuals is shown. (E)TNFprotein concentrationmeasured by

    LuminexintheBCGstimulatedcondition,forLTBIandTB.(F)TNFRSF1BmRNA

    expression levelsmeasuredbyNanostring in theBCGstimulatedcondition, for

    LTBIandTB.(G)TNFgenescoreintheBCGstimulatedconditionfortheLTBIand

    TBgroups.(H)SpearmancorrelationbetweenIL-1ralevelsandTNFgenescore

    intheBCGstimulatedcondition.Paired(A,B,C)andunpaired(E,FandG)non-

    parametric t-test corrected formultiple comparisons. Red line:median values.

    Closedcircle:HealthyControls,Closedsquare:LTBI,Opentriangle:TB.

    Figure5.TBpatientspresentlowergranzymeconcentrationswhichimpact

    levels of functional IL-1α and IL-1β. (A) Levels of IL-1α and IL-1β protein

    measuredbyLuminex intheNullandBCGstimulatedconditions.(B)Levelsof

    IL1AandIL1BmRNAmeasuredbyNanostringintheNullandBCGconditions.(C)

    BCGstimulationofhealthydonorbloodwiththeSykinhibitorR406(5μM)orthe

    preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.17.423082doi: bioRxiv preprint

    https://doi.org/10.1101/2020.12.17.423082

  • 18

    serine-protease inhibitor 3,4-Dichloroisocoumarin (10 μM). Levels of mRNA

    expression for inflammasomecomponentsNLRP3,SYK,CRAD9andCASP1(D),

    cathepsins(E)andgranzymes(F)measuredbyNanostringintheNullandBCG

    conditions for TB and LTBI. (G) Granzyme A and granzyme B protein

    concentrations in TruCultureBCG supernatants for TB and LTBImeasured by

    Luminex.(H)Spearmancorrelationbetweenproteinconcentrationsofgranzyme

    B and IL-1α, and granzyme A and IL-1β in BCG supernatants. Paired (C) and

    unpaired non-parametric t-test corrected for multiple comparisons. Red line:

    median values. Closed square: LTBI, Open triangle: TB, Closed circle: Healthy

    Controls.

    Figure6.IL-1βsignallingisperturbedinactiveTBdiseaseandisresetafter

    successfulantibiotictreatment.(A)HeatmapshowingexpressionlevelsofIL-

    1β-inducedgenes(NullvsIL-1βq=0.001)measuredbyNanostringinLTBIand

    TB,invisit1pre-treatment(Pre-TxV1)andvisit2post-treatment(Post-TxV2).

    (B)LevelsofNFKB1,NFKB2,IRAK2andIRAK3mRNAmeasuredbyNanostring

    afterIL-1βstimulationforLTBIandTB.Unpairednon-parametrict-testcorrected

    formultiple comparisons.Red line:median values. Closed square:LTBI,Open

    triangle:TB.

    MATERIALSANDMETHODS

    Participantgroups

    AdultpatientswithsputumXpertMTB/RIF-positiveTBdiseasewhotestedHIV-

    negative were identified and recruited at clinics in Worcester, South Africa62.

    Healthy QuantiFERON-TB Gold (QFT) positive (M.tb-infected) adults were

    recruited from communities living in or around Worcester and those who

    matchedTBcasesbyageandgenderwereenrolledasLTBIcontrols(Table1).

    BloodwascollectedpriortotreatmentinitiationinactiveTBcasesandagain12-

    18monthslater,aftersuccessfulcompletionoftreatmentdefinedbyclinicalcure

    (V2,n=18).FortheQFT+controls,bloodwasalsocollectedatasecondtime-point,

    12-18monthsaftertheinitialvisit(V2,n=19).TheTBclinicalstudyprotocolsand

    preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.17.423082doi: bioRxiv preprint

    https://doi.org/10.1101/2020.12.17.423082

  • 19

    informedconsentformswereapprovedbytheHumanResearchEthicsCommittee

    of the University of Cape Town (ref: 234/2015). Healthy donor blood was

    obtainedfromtheCoSImmGEncohortoftheInvestigationCliniqueetAccèsaux

    Ressources Biologiques (ICAReB) platform, (Centre de Recherche

    Translationnelle, Institut Pasteur, Paris, France) or from theEtablissement

    francais du sang (EFS, France). Blood was collected in sodium-heparin tubes.

    Writteninformedconsentwasobtainedfromallstudyparticipants.

    TruCultureWholeBloodStimulation

    TruCulturetubeswerepreparedinbatchaspreviouslydescribed14,15,62withthe

    following stimuli: QFT antigens (ESAT-6, CFP-10, TB7.7), Bacillus Calmette–

    Guérin(BCG;SanofiPasteur,105units/ml),andIL-1β(Peprotec,25ng/ml).They

    wereresuspendedin2mlofbufferedmediaandmaintainedat-20°Cuntiluse.

    Blood was collected in sodium-heparin tubes (50 IU/ml final concentration).

    Within30minofcollection,1mlofwholebloodwasdistributedintopre-warmed

    TruCulturetubes,insertedintoadryblockincubator,andmaintainedat37°C(+/-

    1C), roomair for22hr (+/-15min).After incubation, avalvewas inserted to

    separate cells from the supernatantand tostop the stimulationreaction.Upon

    removaloftheliquidsupernatant,cellpelletswereresuspendedin2mlTrizolLS

    (Sigma),vortexedfor2min,andrestedfor10minatroomtemperaturebefore-

    80°Cstorage.Cytokinekineticsecretiondatasetswerepreviouslydescribed33.All

    stimulationswereperformedfor22hunlessotherwisespecified.

    Isolationandcultureofbloodleukocytesfromhealthydonors

    For monocyte experiments, blood was collected in buffy coats from healthy

    donors sampled at the EFS, France. First, PBMCs were isolated by density

    centrifugation from peripheral blood leukocyte separation medium (Ficoll-

    PaqueTM plus; GE Healthcare). Human monocytes were purified by negative

    selectionwith the classicalmonocyte isolation kit (Miltenyi). Monocytes were

    culturedat1x106cells/mLinRPMI1640(Invitrogen,Gaithersburg,MD)(R10)

    containing 10% heat-inactivated fetal bovine serum, penicillin (100 U/ml),

    streptomycin(100μg/ml;1%pen-strep),and1mMglutamine(HyClone,Logan,

    UT).

    preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.17.423082doi: bioRxiv preprint

    https://doi.org/10.1101/2020.12.17.423082

  • 20

    Proteinanalysis

    SupernatantsfromTruCulturetubeswerethawedoniceandtestedbyLuminex

    xMAPtechnologyforatotalof32proteinsincludingcytokines,chemokinesand

    growthfactorsaspreviouslydescribed14.SamplesweremeasuredontheMyriad

    RBMIncplatform(Austen,Texas,US)accordingtoCLIAguidelines(setforthby

    theUSAClinicalandLaboratoryStandardsInstitute).The leastdetectabledose

    (LDD)foreachassaywasderivedbyaveragingthevaluesobtainedfrom200runs

    withthematrixdiluentandadding3standarddeviationstothemean.Thelower

    limitofquantification(LLOQ)wasdeterminedbasedonthestandardcurve for

    eachassayandisthelowestconcentrationofananalyteinasamplethatcanbe

    reliably detected and at which the total error meets CLIA requirements for

    laboratoryaccuracy.IL-1β,IL-1ra,IL-1α,IL-6,IP-10andGranzymeA/Bwerealso

    measuredusingHumanELISAKit(ThermoFischerScientific)orHumanCustom

    Procarta-plexes (ThermoFischer Scientific), following manufacturer’s

    instructions.

    Todetect lowconcentrationsofcytokineshomebrewSimoadigitalELISAwere

    usedforIFNα67andIFNβ68,aspreviouslydescribed.

    Geneexpressionanalysis

    CellpelletsinTrizolLSwerethawedonicefor60minpriortoprocessing.Tubes

    werevortexedtwicefor5minat2000rpmandcentrifuged(3000xgfor5minat

    4°C) topellet thecellulardebrisgeneratedduring lysis.RNAwas isolated from

    wholebloodsamplesusingtheNucleoSpin96RNAtissuekitprotocol(Macherey-

    Nagel)withsomemodificationsaspreviouslydescribed15.

    RNAintegritywasassessed(AgilentRNAkitsforthe2100BioanalyzerSystem).

    TheNanoStringnCountersystemwasusedforthedigitalcountingoftranscripts.

    RNAwasquantifiedusingtheQubitRNAHSAssayKit(ThermoFischerScientific)

    and 100ng of total RNA hybridized with the Human Immunology v2 (plus 30

    additional genes (SupplementaryTable 1) relevant for TB thatwere included)

    GeneExpressionCodeSetaccordingtothemanufacturer’sinstructions.Samples

    wereprocessedin7batches(4forVisit1,3forVisit2),withinwhichthesamples

    were randomized, and the same lot of reagents was used for all samples. All

    preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.17.423082doi: bioRxiv preprint

    https://doi.org/10.1101/2020.12.17.423082

  • 21

    samples were normalized together following background subtraction of the

    negativecontrolprobes,usingpositivecontrolprobesandhousekeepinggenes

    (SDHA,HPRT1,POLR2A,RPL19,G6PD,TBP)selectedbytheGNormmethodas

    previously described69. This was done using the nSolverTM analysis software

    (NanoString technologies). Quality control for our data involved checking the

    followingmetrics:fieldsofviewcounted(flagif<0.75),bindingdensity(flagifnot

    inthe0.05-2.75range),linearityofpositivecontrols(flagifR2<0.9)andlimitof

    detectionforpositivecontrols(flagif0.5fMpositivecontrol<2SDsabovethe

    meanofthenegativecontrols).

    Metabolomicanalysis

    Supernatants fromTruCultureNull tubeswerethawedandtestedbyUltrahigh

    Performance Liquid Chromatography-Tandem Mass Spectroscopy (UPLC-

    MS/MS)foratotalof696metabolites.SamplesweremeasuredontheMetabolon

    Inc platform (Morrisville, North Carolina, US). For metabolite quantification,

    peakswerequantifiedusingarea-under-the-curve.Adatanormalizationstepwas

    performed to correct variation resulting from instrument inter-day tuning

    differences,andvalueswererescaledtosetthemedialequalto1.Missingvalues

    wereimputedwiththeminimum.

    Chemicalinhibitors,agonistsandantagonists

    TheinhibitorofSykphosphorylationR406(Invivogen)andtheserine-protease

    inhibitor3,4-Dichloroisocoumarin(SigmaAldrich)wereusedat5μMand10μM,

    respectively.ThePPARγagonistRosiglitazone(Sigma-Aldrich)andtheantagonist

    GW9662(Sigma-Aldrich)wereusedat10μM.Alldrugswereadded1hbeforeBCG

    stimulation.

    Cytokinestimulationandblockingexperiments

    Wholebloodfromhealthydonorswaspre-incubatedwithantibodiesorarelevant

    isotypecontrolat10µg/mLfor1h(unlessotherwisestated),usingthefollowing:

    anti-humanCD120a(CloneMABTNFR1-B1,BDBioscience)antibody,anti-human

    Interferonalpha/betareceptor1antibody(Anifrolumab),anti-humanInterferon

    alpha/betareceptor2antibody(PBL,cloneMMHAR-2). Wholebloodwasthen

    preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.17.423082doi: bioRxiv preprint

    https://doi.org/10.1101/2020.12.17.423082

  • 22

    diluted1/3inTruCulturemediaandstimulatedwithBCGorwithselectedhuman

    recombinantcytokinesfor22hat37°C,asfollows:IFNα(IntronA;1000IU/mL),

    IFNβ(Betaferon;1000IU/mL),IFNγ(Peprotec;25ng/mL),IL-1β(Peprotech;25

    ng/mL),TNF(Miltenyi;10ng/mL),IL-8(Biolegend;25ng/mL).

    siRNAexperiments

    Monocyteswereseededat2x105cells/200μlin96-wellplatesandincubatedat

    37°C.CyclophilinB(control)andPPARγsiRNA(SMARTPool,Dharmarcon)were

    diluted in DOTAP (1,2-dioleoyl-3-trimethylammonium-propane; Roche Applied

    Sciences).Themixwasgentlymixedandincubatedatroomtemperaturefor15

    min.Afterincubation,themixwasaddedtocellsincultureatafinalconcentration

    of 160 nM. Cellswere then incubated at 37°C for24hours before addingBCG

    stimulationfor16hours.

    Flowcytometry–CD36&PPARγ

    CellswerewashedinPBSandresuspendedinPBScontaining2%fetalcalfserum

    and2mMEDTAandstainedwiththeextracellularmixusinganti-humanCD14-

    BV421 antibody (clone M5E2, BD Bioscience) and anti-human CD36-BUV605

    antibody(cloneCD38,BDBioscience)at1:200.ForPPARγintracellularstaining,

    a Fixation/Permeabilization Solution Kit (BD Cytofix/Cytoperm) was used

    accordingtothemanufacturer’sprotocol.Briefly,thecellswerefixedfor10min

    at4°Cwith100µLoftheFixation/Permeabilizationsolutionandthenwashedand

    stainedin100µLoftheBDPerm/WashBuffercontaininganti-humanpolyclonal

    PPARγantibody(PA3-821A,Invitrogen)at1:25for1hourat4°C.Goatanti-rabbit

    IgG-AlexaFluor700wasusedasasecondaryantibodyat1:1000(ThermoFisher

    Scientific).DataacquisitionwasperformedonaFACSLSRflowcytometerusing

    FACSDiva software (BD Biosciences, San Jose, CA). FlowJo software (Treestar,

    Ashland,OR)wasusedtoanalyzedata.

    StatisticalAnalysis,DataVisualization,andSoftware

    ANOVA(Kruskal-Wallis)testingwasperformedformulti-groupcomparisonsand

    paired t-tests for intra-patient analysis with Qlucore Omics Explorer, v.3.2

    preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.17.423082doi: bioRxiv preprint

    https://doi.org/10.1101/2020.12.17.423082

  • 23

    (Qlucore) or GraphPAD Prism. To correct for multiple testing we report false

    discoveryrate(FDR)-adjustedANOVApvalues;qvalues.Dotplotgraphswere

    compiledwithGraphPadPrismv.6.0,heatmapsandPrincipalComponentAnalysis

    (PCA)plotswithQlucore.ROCcurveswerecalculatedwithR(v.3.4.4)andresults

    drawnwith graphical package ggplot2 (v.3.1.0). UpSet plotswere drawnwith

    UpSetR(v1.3.3),andcorrelationsplotscorrplot(v0.84).

    Time series analysis on the protein secretion measurements, from 5 different

    donors, was conducted using a linear mixed-model approach. The time

    dependencywasmodelledbyincorporatingtheparametertimeasacontinuous

    linear predictor, alongside the other protein predictors, and the donors were

    modelledasarandomeffect.Themodelwas implementedusingtheRpackage

    'lme''(v1.1-20).

    For calculationof cytokine-specific genes score, thegenesuniquely inducedby

    eachcytokinewereidentifiedfromourpreviousstudyinhealthydonors15.The

    firstPrincipalcomponentoftheexpressionmatrixwascomputedusingthePCA()

    functionoftheFactoMineRpackage(version1.42)withtheoptions"scale"setto

    TRUE,andtheoption"ncp"setto1.Positionofeachsampleonthefirstcomponent

    wasthenextractedandusedforfurtheranalysis(Rv.3.6.1).

    Pearson correlations were performed in R (v3.6.0) between IL-1Ra protein

    concentrations quantified by Luminex and the metabolites quantified through

    mass spectrometry for either TB or LTBI cases. To down-size the number of

    metabolitesanalyzed,onlymetabolitesthatbelongtometabolicgroupscomposed

    of at least7metabolitesand thatarenot labelledas ‘Drug’or ‘Chemical’were

    considered.Themetaboliteandproteincorrelationheatmapwasplottedwiththe

    ggplot2Rpackage(v.3.3.1).

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    4. LúciaMoreira-Teixeira,Mayer-Barber,K.,Sher,A.&O’Garra,A.TypeI

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  • 102

    103

    104

    CLE

    C7A

    (tot

    al m

    RN

    A c

    ount

    s) q=1.91 x 10-6

    LTBI TB102

    103

    IRF4

    (tot

    al m

    RN

    A c

    ount

    s)

    q=5.23 x 10-5

    LTBI TB101

    102

    103

    104

    NFK

    B1

    (tot

    al m

    RN

    A c

    ount

    s)

    q=0.0008

    LTBI TB102

    103

    104

    TLR

    8 (t

    otal

    mR

    NA

    cou

    nts)

    q=1.81 x 10-8

    LTBI TB

    D

    F

    C

    E

    A

    B LTBI ≠ TB (q

  • 101

    102

    103

    104

    IL1r

    a (p

    g/m

    l)

    Null BCG

    q=0.0005 q=0.0001

    A

    B DC

    Figure 2

    101 102 103 104 105101

    102

    103

    104

    IL-8 (pg/ml)

    IL-1

    ra p

    g/m

    l

    102

    103

    IL-1

    ra (p

    g/m

    l)

    p=0.8

    Null IL-8

    101

    102

    103

    104

    105

    CX

    CR

    1 m

    RN

    A (t

    otal

    cou

    nts)

    LTBI TB

    q=0.0001

    102

    103

    104

    105

    CX

    CR

    2 m

    RN

    A (t

    otal

    cou

    nts)

    q=0.0002

    LTBI TB101

    102

    103

    104

    105

    IL-8

    (pg/

    ml)

    p=0.0001

    LTBI TB

    E

    F G

    LTBI

    -2 0 2 4 6

    5

    10

    15

    PPARG (RNAseq expression units)

    IL1R

    N

    (RN

    Ase

    q ex

    pres

    sion

    uni

    ts)

    101

    102

    103

    104

    IL1r

    a (p

    g/m

    l)

    p=0.0007

    LTBI TB 101 102 103 104 105101

    102

    103

    104

    IL-8 (pg/ml)

    IL-1

    ra p

    g/m

    l

    r = 0.54 (****)

    101 102 103 104 105101

    102

    103

    104

    IL-8 (pg/ml)

    IL-1

    ra p

    g/m

    l

    Null: LTBIr = 0.4 (ns) TBr = 0.93 (****)

    101

    102

    103

    104

    105

    IL-8

    (pg/

    ml)

    p=0.002 (**)

    LTBI TB

    Gene expression correlation with IL-1ra/IL-8 protein in TB (r>0.7)

    IL-1ra IL-8SPP1IL1RNFER1L3CD44FN1CD36BCAP31PPARGCLEC5AC3

    (Rajaram 2010 - IL-8)

    Unsti

    m2D

    G2F

    DG Eto

    Oligo

    A0

    2000

    4000

    6000

    8000

    CD

    36 g

    MFI

    Monocytes

    q=0.4 (ns)

    q=0.003 (**)

    0

    1000

    2000

    3000

    4000

    CD

    36 m

    RN

    A (t

    otca

    l cou

    nts)

    q=0.0008 (***)

    0

    20

    40

    60

    80

    PPA

    RG

    mR

    NA

    (tot

    cal c

    ount

    s) q=0.0001 (***)

    Null

    How does PPARg get atcivated? - Look for natural ligands —> difference TB vs LTBI? - Upstream options —> PXR (publc datasets?) (Zhou 2008, Wahli 2008)

    Eicosanoid, linoleate, arachidonic LTBI TB

    103

    104

    CD

    44 m

    RN

    A (t

    otca

    l cou

    nts)

    q=0.006 (*)

    100

    101

    102

    103

    104

    FN1

    mR

    NA

    (tot

    cal c

    ount

    s)

    q=0.004 (**)

    100 101 102102

    103

    104

    PPARG mRNA (total counts)

    CD

    36 m

    RN

    A (t

    otal

    cou

    nts) Pearson

    LTBIr = 0.6 (**)TBr = 0.3 (ns)Total = 0.51 (***)

    SpearmanLTBIr = 0.53 (**)TBr = 0.18 (ns)Total = 0.55 (****)

    Gene expression correlation with IL-1ra/IL-8 protein in TB (r>0.7)

    IL-1ra IL-8 PPARGCD36CD44FN1SPP1IL1RNFER1L3BCAP31CLEC5AC3

    Gene expression correlation with IL-1ra/IL-8 protein in TB (r>0.7)

    Infected hMDM

    Uninfected (r=0.73 q=0.02)Pathogenic mycobacteria (r=0.34 q=0.03)Non-mycobacteria (r=-0.28 q=0.1)

    H

    HCTB

    TB LTBI

    10-1

    100

    101

    102

    103

    IL1a

    (pg/

    ml)

    Null BCG

    q=0.1 q=0.0001

    100

    101

    102

    103

    104

    IL1b

    (pg/

    ml)

    Null BCG

    q=0.1 q=0.0001

    101

    102

    103

    104

    IL1r

    a (p

    g/m

    l)

    Null BCG

    q=0.0005 q=0.0001

    0.00

    0.05

    0.10

    0.15

    IL1a

    /IL1r

    a

    q=0.0001

    LTBI TB

    0

    2

    4

    6

    8

    10

    IL1b

    /IL1r

    a

    q=0.0001

    LTBI TB

    100

    101

    102

    103

    104

    IL1A

    mR

    NA

    t(ota

    l cou

    ntsl

    )

    Null BCG

    q=0.6 q=0.2

    101

    102

    103

    104

    105

    IL1B

    mR

    NA

    (tota

    l cou

    nts)

    Null BCG

    q=0.9 q=0.3

    101

    102

    103

    104

    IL1R

    N m

    RN

    A (to

    tal c

    ount

    sl)

    Null BCG

    q=0.0004 q=0.4

    A

    B

    D FE

    G

    C

    H

    θ θ

    R406

    5 µM

    DCI 1

    0 µM

    101

    102

    103

    104

    IL1b

    (pg/

    ml)

    q=0.02

    q=0.02

    I

    101

    102

    103

    Gra

    nzym

    e B

    (pg/

    ml)

    p= 0.03

    LTBI TB

    J

    101 102 103100

    101

    102

    103

    Granzyme B (pg/ml)

    IL1a

    (pg/

    ml)

    101 102 103102

    103

    104

    Granzyme B (pg/ml)

    IL1b

    (pg/

    ml)

    r = 0.53 p

  • 5α-pregnane-3β,20α-diol monosulfate (2)5α-pregnane-3β,20α-diol monosulfate (1)5α-pregnane-3β,20α-diol disulfatePregnanediol-2-glucoronideSphingomyelin (d18:1/20:2, d18:2/20:1, d16:1/22:2)5α-pregnane-3(α/β),20β-diol disulfate

    BLTBIr = 0.02 (q=0.9)TBr = 0.55 (q=0.01)

    Figure 3

    10-1 100 101 102101

    102

    103

    104

    5α-pregnane-3β,20α-diol disulfate

    IL1r

    a (p

    g/m

    l)

    A

    C

    LTBI

    D

    101

    102

    103

    104

    105

    IL-1

    ra (p

    g/m

    l)

    q=0.008q=0.008

    NullBCG

    siCTL siPPARG

    E

    0

    2

    4

    6

    Rat

    io IL

    -1ra

    BC

    G/N

    ull

    siCTL siPPARG

    p=0.8

    HCTB

    TB

    LTBI

    Phenylalanine and Tyrosine Metabolism.48408Food Component/Plant.36649Glutamate Metabolism.39577Food Component/Plant.48569Leucine, Isoleucine and Valine Metabolism.1591Leucine, Isoleucine and Valine Metabolism.1587Glutamate Metabolism.33943Gamma−glutamyl Amino Acid.33364Phenylalanine and Tyrosine Metabolism.32197Fatty Acid, Monohydroxy.22036Tryptophan Metabolism.18349Gamma−glutamyl Amino Acid.18369Gamma−glutamyl Amino Acid.2734Food Component/Plant.48141Leucine, Isoleucine and Valine Metabolism.33967Gamma−glutamyl Amino Acid.33934Gamma−glutamyl Amino Acid.43829Fatty Acid, Monohydroxy.37752Lysolipid.19260Methionine, Cysteine, SAM and Taurine Metabolism.1868Histidine Metabolism.15716Food Component/Plant.41494Leucine, Isoleucine and Valine Metabolism.1649Tryptophan Metabolism.54Urea cycle; Arginine and Proline Metabolism.33953Food Component/Plant.36095Xanthine Metabolism.34401Histidine Metabolism.607Glutamate Metabolism.46225Xanthine Metabolism.32445Urea cycle; Arginine and Proline Metabolism.32306Glutamate Metabolism.15720Phenylalanine and Tyrosine Metabolism.33950Phospholipid Metabolism.15990Phospholipid Metabolism.37455Methionine, Cysteine, SAM and Taurine Metabolism.2829Gamma−glutamyl Amino Acid.34456Histidine Metabolism.33946Xanthine Metabolism.18392Tryptophan Metabolism.1417Glutamate Metabolism.57Glycine, Serine and Threonine Metabolism.1284Steroid.37192Lysolipid.54885Glutamate Metabolism.53Xanthine Metabolism.34400Phospholipid Metabolism.34396TCA Cycle.1303Fatty Acid, Dicarboxylate.37253Steroid.1712Xanthine Metabolism.18254Urea cycle; Arginine and Proline Metabolism.22137Steroid.37186Glycine, Serine and Threonine Metabolism.37076Phenylalanine and Tyrosine Metabolism.32390Gamma−glutamyl Amino Acid.55015Urea cycle; Arginine and Proline Metabolism.36808Xanthine Metabolism.34404Lysolipid.36600Phospholipid Metabolism.52603Histidine Metabolism.30460Phospholipid Metabolism.52452Sphingolipid Metabolism.57372Xanthine Metabolism.34389Methionine, Cysteine, SAM and Taurine Metabolism.42382TCA Cycle.52282Leucine, Isoleucine and Valine Metabolism.22132Sphingolipid Metabolism.57370Urea cycle; Arginine and Proline Metabolism.1670Gamma−glutamyl Amino Acid.18245Benzoate Metabolism.39600Leucine, Isoleucine and Valine Metabolism.15676Fatty Acid, Dicarboxylate.21134Benzoate Metabolism.48460Sphingolipid Metabolism.53013TCA Cycle.1564Sphingolipid Metabolism.57330Steroid.37207Sphingolipid Metabolism.48492Phenylalanine and Tyrosine Metabolism.1669TCA Cycle.1437Lysolipid.35186Methionine, Cysteine, SAM and Taurine Metabolism.39592Histidine Metabolism.43255Fatty Acid, Dicarboxylate.36754TCA Cycle.1643Fatty Acid, Monohydroxy.42489Glycine, Serine and Threonine Metabolism.1516Phenylalanine and Tyrosine Metabolism.53242Methionine, Cysteine, SAM and Taurine Metabolism.590Lysolipid.34214Steroid.37203Methionine, Cysteine, SAM and Taurine Metabolism.2125Phenylalanine and Tyrosine Metabolism.64Leucine, Isoleucine and Valine Metabolism.44526Fatty Acid Metabolism(Acyl Carnitine).57516Steroid.37187Fatty Acid, Monohydroxy.39609Leucine, Isoleucine and Valine Metabolism.60Phenylalanine and Tyrosine Metabolism.1299Fatty Acid, Monohydroxy.21239Lysolipid.36594Fatty Acid, Dicarboxylate.20676Fatty Acid, Monohydroxy.32457Secondary Bile Acid Metabolism.42574Phospholipid Metabolism.40406Fatty Acid Metabolism(Acyl Carnitine).34409Gamma−glutamyl Amino Acid.33422Fatty Acid, Dicarboxylate.18362Leucine, Isoleucine and Valine Metabolism.35107Benzoate Metabolism.35320Xanthine Metabolism.18394Steroid.37184Long Chain Fatty Acid.52674Plasmalogen.52689Fatty Acid, Dicarboxylate.39831Phenylalanine and Tyrosine Metabolism.36845Tryptophan Metabolism.48782Long Chain Fatty Acid.12125Sphingolipid Metabolism.52605Fatty Acid Metabolism(Acyl Carnitine).57520Methionine, Cysteine, SAM and Taurine Metabolism.43378Steroid.47112Food Component/Plant.18335Methionine, Cysteine, SAM and Taurine Metabolism.1589Tryptophan Metabolism.27513Histidine Metabolism.43493Xanthine Metabolism.569Sphingolipid Metabolism.47154Fatty Acid, Dicarboxylate.39837Glycine, Serine and Threonine Metabolism.5086Tryptophan Metabolism.33959Xanthine Metabolism.39598Polyunsaturated Fatty Acid (n3 and n6).37478Secondary Bile Acid Metabolism.34093Benzoate Metabolism.46165Steroid.37202Phospholipid Metabolism.52468Plasmalogen.52677Benzoate Metabolism.46111Methionine, Cysteine, SAM and Taurine Metabolism.48187Steroid.31591Urea cycle; Arginine and Proline Metabolism.15497Steroid.32562Lysolipid.35628Fatty Acid Metabolism(Acyl Carnitine).57517Fatty Acid Metabolism(Acyl Carnitine).57529Long Chain Fatty Acid.32418Phospholipid Metabolism.53176Steroid.37210Plasmalogen.52716Food Component/Plant.48715Lysolipid.34419Methionine, Cysteine, SAM and Taurine Metabolism.22176Benzoate Metabolism.48429Urea cycle; Arginine and Proline Metabolism.32984Sphingolipid Metabolism.52436Fatty Acid Metabolism(Acyl Carnitine).57513Fatty Acid Metabolism(Acyl Carnitine).46223Phospholipid Metabolism.52710Fatty Acid Metabolism(Acyl Carnitine).57515Xanthine Metabolism.32391Diacylglycerol.54991Steroid.37211Steroid.32619Steroid.37190Glycine, Serine and Threonine Metabolism.33939Histidine Metabolism.32350Long Chain Fatty Acid.1358Monoacylglycerol.21232Fatty Acid, Monohydroxy.22053Steroid.1769Steroid.33973Phospholipid Metabolism.52616Sphingolipid Metabolism.47153Medium Chain Fatty Acid.32492Phenylalanine and Tyrosine Metabolism.2761Xanthine Metabolism.34424Polyunsaturated Fatty Acid (n3 and n6).33969Phenylalanine and Tyrosine Metabolism.48407Methionine, Cysteine, SAM and Taurine Metabolism.1302Sphingolipid Metabolism.48493Long Chain Fatty Acid.1552Food Component/Plant.31536Glutamate Metabolism.54923Phospholipid Metabolism.52447Leucine, Isoleucine and Valine Metabolism.22116Phenylalanine and Tyrosine Metabolism.48433Phospholipid Metabolism.42446Steroid.32827Glutamate Metabolism.42370Medium Chain Fatty Acid.33968Glycine, Serine and Threonine Metabolism.1648Fatty Acid Metabolism(Acyl Carnitine).57528Sphingolipid Metabolism.57478Lysolipid.36602Polyunsaturated Fatty Acid (n3 and n6).35718Leucine, Isoleucine and Valine Metabolism.36746Urea cycle; Arginine and Proline Metabolism.1493Histidine Metabolism.40730Fatty Acid Metabolism(Acyl Carnitine).57519Lysolipid.19324Polyunsaturated Fatty Acid (n3 and n6).32415Plasmalogen.52475Fatty Acid Metabolism(Acyl Carnitine).32198Long Chain Fatty Acid.1118Benzoate Metabolism.18281Glycine, Serine and Threonine Metabolism.58Phenylalanine and Tyrosine Metabolism.42040Fatty Acid, Monohydroxy.17945Gamma−glutamyl Amino Acid.33947Plasmalogen.52682Sphingolipid Metabolism.34445Diacylglycerol.54943Plasmalogen.52478Histidine Metabolism.59Sphingolipid Metabolism.52435Secondary Bile Acid Metabolism.18477Phenylalanine and Tyrosine Metabolism.32553Benzoate Metabolism.15753Fatty Acid Metabolism(Acyl Carnitine).33936Food Component/Plant.38637Fatty Acid Metabolism(Acyl Carnitine).57512Phospholipid Metabolism.19130Polyunsaturated Fatty Acid (n3 and n6).34035Fatty Acid, Monohydroxy.52938Steroid.37209TCA Cycle.528Urea cycle; Arginine and Proline Metabolism.57461Long Chain Fatty Acid.33587Phospholipid Metabolism.42450Long Chain Fatty Acid.52285Phenylalanine and Tyrosine Metabolism.22130Long Chain Fatty Acid.1365Fatty Acid Metabolism(Acyl Carnitine).57518Fatty Acid Metabolism(Acyl Carnitine).35160Plasmalogen.52673Sphingolipid Metabolism.52604Sphingolipid Metabolism.17747Lysolipid.45951Glycine, Serine and Threonine Metabolism.27710Fatty Acid, Dicarboxylate.35678Leucine, Isoleucine and Valine Metabolism.15745Sphingolipid Metabolism.54979Urea cycle; Arginine and Proline Metabolism.35127Secondary Bile Acid Metabolism.12261Phospholipid Metabolism.52449Phospholipid Metabolism.52467Diacylglycerol.54942Methionine, Cysteine, SAM and Taurine Metabolism.44878Phospholipid Metabolism.1600Lysolipid.45966Monoacylglycerol.48341Sphingolipid Metabolism.52434Benzoate Metabolism.36847TCA Cycle.52304Long Chain Fatty Acid.33447Fatty Acid Metabolism(Acyl Carnitine).57521Fatty Acid Metabolism(Acyl Carnitine).33941Benzoate Metabolism.48763Food Component/Plant.37459Medium Chain Fatty Acid.1644Sphingolipid Metabolism.44877Long Chain Fatty Acid.33972Secondary Bile Acid Metabolism.32807Diacylglycerol.57449Benzoate Metabolism.36098Lysolipid.33961Phospholipid Metabolism.52438Secondary Bile Acid Metabolism.52975Phospholipid Metabolism.42448Plasmalogen.52477Long Chain Fatty Acid.1356Gamma−glutamyl Amino Acid.44872Diacylglycerol.54945Polyunsaturated Fatty Acid (n3 and n6).1110Urea cycle; Arginine and Proline Metabolism.34387Diacylglycerol.54990Methionine, Cysteine, SAM and Taurine Metabolism.52281Fatty Acid, Monohydroxy.53034Fatty Acid Metabolism(Acyl Carnitine).44681Steroid.38168Fatty Acid, Monohydroxy.35675Phospholipid Metabolism.47155Leucine, Isoleucine and Valine Metabolism.1549Fatty Acid Metabolism(Acyl Carnitine).34534Fatty Acid, Monohydroxy.52916Plasmalogen.52713Diacylglycerol.54968Secondary Bile Acid Metabolism.36850Phenylalanine and Tyrosine Metabolism.1432Fatty Acid Metabolism(Acyl Carnitine).38178Diacylglycerol.52634Sphingolipid Metabolism.48490Sphingolipid Metabolism.53010Steroid.32425Leucine, Isoleucine and Valine Metabolism.15765Methionine, Cysteine, SAM and Taurine Metabolism.42374Food Component/Plant.598Phospholipid Metabolism.19263Secondary Bile Acid Metabolism.1605Polyunsaturated Fatty Acid (n3 and n6).18467Tryptophan Metabolism.2342Food Component/Plant.22206Phospholipid Metabolism.52446Medium Chain Fatty Acid.32497Long Chain Fatty Acid.1336Plasmalogen.52748Fatty Acid, Monohydroxy.22001Long Chain Fatty Acid.1121Fatty Acid Metabolism(Acyl Carnitine).32328Lysolipid.48258Long Chain Fatty Acid.33971Sphingolipid Metabolism.17769Fatty Acid Metabolism(Acyl Carnitine).57511Phenylalanine and Tyrosine Metabolism.1567Leucine, Isoleucine and Valine Metabolism.1125Gamma−glutamyl Amino Acid.33949Diacylglycerol.57373Food Component/Plant.57591Fatty Acid, Dicarboxylate.32388Sphingolipid Metabolism.52495Food Component/Plant.38276Histidine Metabolism.32349Diacylglycerol.46799Diacylglycerol.57406Urea cycle; Arginine and Proline Metabolism.1638Monoacylglycerol.32506Sphingolipid Metabolism.52437Diacylglycerol.52633Steroid.38170Phospholipid Metabolism.52684Fatty Acid Metabolism(Acyl Carnitine).43264Plasmalogen.52614Phenylalanine and Tyrosine Metabolism.566Histidine Metabolism.15677Diacylglycerol.57450Medium Chain Fatty Acid.1642Diacylglycerol.46798Tryptophan Metabolism.48757Phospholipid Metabolism.52462Diacylglycerol.54957Steroid.46115Food Component/Plant.48674Tryptophan Metabolism.1512Phospholipid Metabolism.42449Urea cycle; Arginine and Proline Metabolism.1898Polyunsaturated Fatty Acid (n3 and n6).1105Lysolipid.42398Monoacylglycerol.33419Lysolipid.52690Phenylalanine and Tyrosine Metabolism.12017Secondary Bile Acid Metabolism.39379Secondary Bile Acid Metabolism.32620Diacylglycerol.54946Tryptophan Metabolism.27672Phospholipid Metabolism.52683Glycine, Serine and Threonine Metabolism.3141Phenylalanine and Tyrosine Metabolism.48841Food Component/Plant.21151Leucine, Isoleucine and Valine Metabolism.45095Food Component/Plant.33935Medium Chain Fatty Acid.12035Polyunsaturated Fatty Acid (n3 and n6).17805Phospholipid Metabolism.52461Monoacylglycerol.27447Sphingolipid Metabolism.57475Sphingolipid Metabolism.57480Diacylglycerol.54953Urea cycle; Arginine and Proline Metabolism.2132Sphingolipid Metabolism.42459Sphingolipid Metabolism.57331Polyunsaturated Fatty Acid (n3 and n6).44675Phospholipid Metabolism.52726Diacylglycerol.54954Lysolipid.35631Food Component/Plant.20699Methionine, Cysteine, SAM and Taurine Metabolism.18374Sphingolipid Metabolism.52234TCA Cycle.37058Sphingolipid Metabolism.42463Fatty Acid Metabolism(Acyl Carnitine).53223Sphingolipid Metabolism.52433Monoacylglycerol.21184Sphingolipid Metabolism.48491Diacylglycerol.54963Fatty Acid Metabolism(Acyl Carnitine).48182Diacylglycerol.54958Lysolipid.41220Fatty Acid, Dicarboxylate.31787Secondary Bile Acid Metabolism.57577Fatty Acid Metabolism(Acyl Carnitine).33952Diacylglycerol.54964Diacylglycerol.57364Leucine, Isoleucine and Valine Metabolism.33937Diacylglycerol.54956Phospholipid Metabolism.52464Benzoate Metabolism.35527Phospholipid Metabolism.52470Leucine, Isoleucine and Valine Metabolism.32397Sphingolipid Metabolism.37506Tryptophan Metabolism.15140Lysolipid.35305Diacylglycerol.55001Sphingolipid Metabolism.57365Diacylglycerol.54966Phenylalanine and Tyrosine Metabolism.18280Monoacylglycerol.21127Diacylglycerol.54965Fatty Acid, Monohydroxy.53230Long Chain Fatty Acid.1361Lysolipid.33228TCA Cycle.12110Urea cycle; Arginine and Proline Metabolism.55072Polyunsaturated Fatty Acid (n3 and n6).32504Fatty Acid, Dicarboxylate.42395Gamma−glutamyl Amino Acid.37063Lysolipid.33230Food Component/Plant.37181Lysolipid.36812Gamma−glutamyl Amino Acid.2730Lysolipid.33955Phospholipid Metabolism.15506Leucine, Isoleucine and Valine Metabolism.34407Fatty Acid, Dicarboxylate.32398Phenylalanine and Tyrosine Metabolism.15749Monoacylglycerol.35625Sphingolipid Metabolism.57474Benzoate Metabolism.15778Sphingolipid Metabolism.52615Diacylglycerol.54967Lysolipid.35253Tryptophan Metabolism.32405Sphingolipid Metabolism.57479Medium Chain Fatty Acid.12067Sphingolipid Metabolism.57482Secondary Bile Acid Metabolism.32599Phospholipid Metabolism.52450Food Component/Plant.587Diacylglycerol.54950Food Component/Plant.46144Diacylglycerol.54955Lysolipid.45970Diacylglycerol.54961Fatty Acid, Dicarboxylate.35669Lysolipid.47118Phospholipid Metabolism.52669Food Component/Plant.34384Diacylglycerol.54960Lysolipid.34428Monoacylglycerol.19266Gamma−glutamyl Amino Acid.36738Phenylalanine and Tyrosine Metabolism.36103Lysolipid.46325Leucine, Isoleucine and Valine Metabolism.33441Benzoate Metabolism.46146Urea cycle; Arginine and Proline Metabolism.37431Sphingolipid Metabolism.57473Food Component/Plant.38100Monoacylglycerol.35153Monoacylglycerol.34397Monoacylglycerol.34393Diacylglycerol.54949Sphingolipid Metabolism.37529Tryptophan Metabolism.38116Methionine, Cysteine, SAM and Taurine Metabolism.56Urea cycle; Arginine and Proline Metabolism.43249Medium Chain Fatty Acid.1645Monoacylglycerol.53220Fatty Acid Metabolism(Acyl Carnitine).52984Sphingolipid Metabolism.57477Tryptophan Metabolism.37097Food Component/Plant.48428Monoacylglycerol.52431Sphingolipid Metabolism.19503Benzoate Metabolism.36099Steroid.46172Sphingolipid Metabolism.57481Steroid.40708Steroid.37198Steroid.37196Steroid.37200

    −0.6 −0.2 0.2 0.6Value

    050

    100

    150

    Color Keyand Histogram

    Cou

    nt 103

    104

    IL-1

    ra (p

    g/m

    l)

    q=0.0002 q=0.04

    / Rosi

    BCG

    /GW9662

    preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.17.423082doi: bioRxiv preprint

    https://doi.org/10.1101/2020.12.17.423082

  • A

    G H

    0 10 20 30

    10-5

    100

    105

    Time post BCG stimulation (h)

    pg/m

    l IL1raIFNα (q=0.6)IFNβ (q=0.6)TNF (q=0.002)

    C

    Figure 4

    B

    0

    2

    4

    6

    8

    TNF

    scor

    eq=0.002

    LTBI TB102

    103

    104

    105

    TNF

    (pg/

    ml)

    q=0.002

    LTBI TB103

    104

    105

    TNFR

    SF1B

    mR

    NA

    (tota

    l cou

    nts) q=0.0001

    LTBI TB

    F

    -5 0 5102

    103

    104

    TNF scoreIL

    1ra

    (pg/

    ml)

    r = 0.32p = 0.03

    BCG

    Null

    TNFIL-

    1βIFNγ

    IFNβ

    IFNα

    IL-8

    101

    102

    103

    104

    105

    IL-1

    ra (p

    g/m

    L)

    q=0.0001

    q=0.0001

    E

    BCG BCG BCG

    D

    Null

    TNF

    IFNα

    IFNβ

    TNF +

    IFNα

    TNF +

    IFNβ

    101

    102

    103

    104

    105

    IL-1

    ra (p

    g/m

    L)

    q=0.03

    q=0.02

    q=0.06

    q=0.01

    /

    Isotyp

    e C

    anti-T

    NFR

    anti-I

    FNAR

    1

    anti-I

    FNAR

    2103

    104

    IL1r

    a (p

    g/m

    l)

    BCG

    q=0.001

    preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.17.423082doi: bioRxiv preprint

    https://doi.org/10.1101/2020.12.17.423082

  • D

    F G

    θ θ

    R406

    5 µM

    DCI 1

    0 µM

    101

    102

    103

    104

    IL1b

    (pg/

    ml)

    q=0.02

    q=0.02

    H

    101

    102

    103

    Gra

    nzym

    e B

    (pg/

    ml)

    q= 0.003

    LTBI TB

    101 102 103100

    101

    102

    103

    Granzyme B (pg/ml)

    IL1a

    (pg/

    ml)

    10-1

    100

    101

    102

    103

    Gra

    nzym

    e A

    (pg/

    ml)

    q= 0.4

    LTBI TB

    10-1 100 101 102 103102

    103

    104

    Granzyme A (pg/ml)

    IL1b

    (pg/

    ml)

    r = 0.33 p=0.02

    r = 0.53 p

  • Figure 6

    Pre-Tx (V1)

    LTBI TB

    Post-Tx (V2)

    101

    102

    103

    104

    NFK

    B1

    mR

    NA

    (tot

    al c

    ount

    s)

    Pre-Tx (V1)

    Post-Tx (V2)

    q=2.79x10-9 q=0.3

    102

    103

    104

    NFK

    B2

    mR

    NA

    (tot

    al c

    ount

    s)

    Pre-Tx (V1)

    Post-Tx (V2)

    q=1.9x10-9 q=0.3