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