LRH-1 mediates anti-inflammatory and antifungal phenotype of IL-13-activated macrophages

13 Pages·2015·1.84 MB·English

by  Lise Lefèvre

ARTICLE
Received30Jan 2015 |Accepted 27 Feb 2015 |Published 15 Apr2015 DOI:10.1038/ncomms7801 OPEN
LRH-1 mediates anti-inflammatory and antifungal
phenotype of IL-13-activated macrophages through
the PPARg ligand synthesis
Lise Lefe`vre1,2, He´le`ne Authier1,2, Sokrates Stein3, Clarisse Majorel2, Bettina Couderc4, Christophe Dardenne1,2,
Mohamad Ala Eddine2, Etienne Meunier1,2, Jose´ Bernad1,2, Alexis Valentin2, Bernard Pipy1,2,*,
Kristina Schoonjans3,* & Agne`s Coste1,2,*
Liver receptor homologue-1 (LRH-1) is a nuclear receptor involved in the repression of
inflammatoryprocessesinthehepatointestinaltract.HerewereportthatLRH-1isexpressed
in macrophages and induced by the Th2 cytokine IL-13 via a mechanism involving STAT6.
Weshowthatloss-of-functionofLRH-1inmacrophagesimpedesIL-13-inducedmacrophage
polarization due to impaired generation of 15-HETE PPARg ligands. The incapacity to
generate 15-HETE metabolites is at least partially caused by the compromised regulation of
CYP1A1 and CYP1B1. Mice with LRH-1-deficient macrophages are, furthermore, highly
susceptible to gastrointestinal and systemic Candida albicans infection. Altogether, these
results identify LRH-1 as a critical component of the anti-inflammatory and fungicidal
response of alternatively activated macrophages that acts upstream from the IL-13-induced
15-HETE/PPARg axis.
1UMRMD3,EA2405PolarisationdesMacrophagesetRe´cepteursNucle´airesdanslesPathologiesInflammatoiresetInfectieuses,UPS,Toulouse31400,
France.2Universite´deToulouse,UMR152,UPS,Toulouse31400,France.3MetabolicSignaling,InstituteofBioengineering,EcolePolytechniqueFe´de´ralede
Lausanne,Lausanne1015,Switzerland.4EA4553IndividualisationdestraitementsdescancersovariensetORL,UPS,Toulouse31400,France.*These
authorscontributedequallytothiswork.CorrespondenceandrequestsformaterialsshouldbeaddressedtoA.C.(email:[email protected]).
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M
acrophages orchestrate innate immune responses by documenting the anti-inflammatory properties of LRH-1 in the
initiating and resolving inflammatory signalling pro- liverandgut,nostudiessofarhavefocusedontheroleofLRH-1in
grammes.Emergingevidenceindicatesthatthestateof macrophages.
macrophage polarization plays a critical role in the regulation of In the present study, we identify LRH-1 as an important
these inflammatory processes. Two different programmes of regulator of the inflammatory response in macrophages. We
macrophage activation, the classical (M1) and the alternative demonstrate that LRH-1 is induced by IL-13 via a STAT6-
differentiation, classify polarized macrophages with either dependent mechanism, which in turn induces the transcriptional
persistence or resolution of inflammation1–3. M1 macrophages activation of CYP1A1 and CYP1B1, two enzymes involved in the
express high levels of opsonic receptors, involved in the generationof15-HETEPPARgligand.Finally,wealsodemonstrate
production of pro-inflammatory effector molecules such as theimportanceofintactLRH-1signallingintheanti-inflammatory
reactive oxygen and nitrogen intermediates and pro- and antifungal functions of alternatively activated macrophages,
inflammatory cytokines (interleukin (IL)-1b, tumour-necrosis indicatingthatmodulatorsofLRH-1activitymayhavetherapeutic
factor alpha (TNFa), IL-6 and IL-12). These macrophages potential torestraininfectious and inflammatory diseases.
contribute to inflammation, microbial killing, regulation of cell
proliferation and apoptosis. Alternatively activated macrophages
are characterized by abundant levels of the anti-inflammatory Results
cytokine IL-10 and non-opsonic receptors, such as C-type lectin IL-13-mediatedLRH-1geneexpressionisdependentonSTAT6.
receptors and scavenger receptors (CD36), and resolve The anti-inflammatory properties of LRH-1 are well established
inflammation by increasing CD36-mediated efferocytosis and in the liver and gut24. To elucidate whether LRH-1 also
secretion of tissue remodelling/repair mediators3,4. participates in regulating the inflammatory response in
The balance of macrophage differentiation in favour of macrophages, gene expression profiling was performed. In situ
alternativelyactivatedmacrophagescanbeshiftedbytheactivation hybridization and reverse transcriptase–quantitative PCR
of the nuclear receptor peroxisome proliferator-activated receptor (RT–qPCR) revealed that LRH-1 (encoded by the Nr5a2 gene),
gamma (PPARg) (refs 5,6). PPARg expression and activity in knowntobeexpressedinthecolonandliver,isalsoexpressedin
macrophages is negatively regulated during inflammatory macrophages but not in B and T immune cells (Fig. 1a).
processes7,8. In addition, activated PPARg transrepresses many Consistentwiththegeneexpressiondata,LRH-1proteinwasalso
inflammation-activated transcription factors, including nuclear detected in macrophages (Supplementary Fig. 1b). We next
factor-kappaB (NF-kB), signal transducers and activators of analysed the impact of pro- and anti-inflammatory factors on
transcription (STATs), activator protein 1 (AP1) and nuclear Nr5a2 gene expression in primary macrophages. As depicted in
factor of activated T-cells NFAT), resulting in pro-inflammatory Fig. 1b, pro-inflammatory challenges, such LPS and IFNg
mediator inhibition9. PPARg is activated by endogenous ligands exposure, but not IL-6, significantly reduced or abolished Nr5a2
derived from the metabolism of arachidonic acid (AA)9. Among mRNA expression. Conversely, IL-13, IL-4 and IL-10 cytokines
these ligands, 15-deoxy-D12,14PGJ (15d-PGJ2), metabolized significantly enhanced Nr5a2 mRNA level in macrophages.
2
through the COX1/COX2 cyclooxygenases, and the 12- and 15- Similar to findings in the murine model, NR5A2 mRNA levels
hydroxyeicosatrienoic acids (HETEs), metabolized through 5 and were significantly increased by IL-13 treatment in human
12/15 lipoxygenases, are essential for PPARg endogenous monocytes (Fig. 1c). These results suggest that LRH-1 could be
activation5,10,11. Inaddition to cyclooxygenases and lipoxygenases, part of the transcriptional network mediating alternative
cytochromeP450(CYP)enzymesarealsoconsideredtobecritical activation of macrophages. To test this hypothesis, we analysed
forthemetabolismofAAinepoxy(EETs)andinhydroxy(HETEs) the downstream signalling components of IL-13 in more detail
derivatives10,11.WithintheCYPfamily,theCYP1familyismainly (Fig. 1d–i). STAT6, a transcription factor known to be activated
involvedinthegenerationof12-and15-HETEsthroughCYP1A1 byIL-13ispartofthesignallingpathwaythatgovernsalternative
and CYP1B1 (refs 12,13). activation25.Interestingly,exposureofmacrophageswithAG490,
Liver receptor homologue-1 (LRH-1, NR5A2) is a nuclear aJak-2/STAT6inhibitor,preventedtheIL-13-mediatedinduction
receptor highly expressed in the intestine, liver, pancreas and of LRH-1 (Fig. 1d). Consistent with these observations, IL-13
ovary14,15. Although LRH-1 has been recognized as an orphan failed to increase Nr5a2 mRNA and protein levels in
receptor,phospholipids,includingthephosphatidylinositolsecond macrophages deficient for STAT6 (Fig. 1e,f), suggesting that
messengers, and more recently the 12C-fatty acyl-containing STAT6 mediates the transcriptional regulation of LRH-1. We
phospholipid, dilauroyl phosphatidylcholine (DLPC), have been then performed transient transfection assays in primary
described to bind the ligand-binding pocket and to macrophages to assess the effect of IL-13 and STAT6 on Nr5a2
act as LRH-1 agonists16–18. LRH-1 plays important roles promoter activity. While 4h of IL-13 exposure was already
in embryonic development, cholesterol and bile acid sufficient to induce Nr5a2 promoter activity in wild-type
homeostasis14,15 and promotes hepatic glucose sensing through macrophages (Fig. 1g,h), chemical inhibition of STAT6 by
theregulationoftheglucokinaseenzyme19.Severallinesofevidence AG490 (Fig. 1g) or genetic deletion of STAT6 (Fig. 1h)
also support a role for LRH-1 in the control of the inflammatory attenuated or even abolished this response.
response. While pro-inflammatory factors such as TNFa and To evaluate whether LRH-1issubject to direct transcriptional
lipopolysaccharide (LPS) decrease LRH-1 expression in murine controlbySTAT6,weperformedaninsilicoanalysisoftheNr5a2
modelsofhumancolontumorigenesis,deficiencyofLRH-1inthe promoter region. Scanning of the Nr5a2 promoter sequence for
intestinalepitheliumpredisposesmicetointestinalinflammationas the STAT6 response element (STAT6-RE) canonical motif
aresultofadefectinlocalglucocorticoidproduction.Inthecolon revealed four putative STAT6-RE (Fig. 1i). Chromatin immuno-
from patients with inflammatory bowel disease, inflammation is precipitation(ChIP)analysisofmacrophageDNAfromC57BL/6
inversely correlated with the expression of LRH-1 (refs 20,21). In mice revealed specific recruitment of STAT6 to site 1 at (cid:2)541,
line with these reports, Venteclef et al.22,23 identified a role for which is most proximal to the transcription initiation site of the
LRH-1 in the negative modulation of the hepatic acute-phase gene (Fig. 1i).
response by inhibiting IL-6- and IL-1b-stimulated haptoglobin, To explore the functionality of this site, we next modified by
serumamyloidAgeneexpressioninhepatocytesandinducinganti- in vitro mutagenesis its sequence and we evaluated the mutated
inflammatory IL-1ra expression. Despite the numerous studies Nr5a2 reporter construct activity on IL-13 exposure (Fig. 1j).
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In situ hybridization
NR5A2 Nr5a2
Relative mRNAexpression level 10..551 Nr5a2 Mφ Relative mRNAexpression level 120...21555 Nr5a2 ** ** * * Relative mRNAexpression level 1243 * Relative mRNAexpression level 21 ** ##
**
0 0 0 0
Colon Liver Mφ LB LT C LPS IFNγ IL-6 IL-13 IL-4 IL-10 IL-13 – + IL-13 – + +
AG490 – – +
Nr5a2 LRH-1-luc LRH-1-luc
Relative mRNAexpression level 1234 ** ##SStt#aa*#*tt66+–//–+ LRTHIBL--P113 –Stat6+/++ –Stat6–/+– k36D81 Fold inductionuciferase activity 1032....1235555 ** #*#* Fold inductionuciferase activity 120...21555 ** SSttaa#tt#66+–//+–
0 l l
0 0
IL-13 – + – +
IL-13 – + – + IL-13 – + – +
+ AG490
Nr5a2 mt
Nr5a2
Site 4 Site 3 Site 2 Site 1
LRH-1-luc –550 Site 1 –532
6 Nr5a2 wt
–3024 –2s7ItP8a0 t:6 Inp–u2t010 sItPa t:6 Inp–u5t41 ductione activity 345 ** SSttaatt66+–//–+
230000 bbpp IL-13 – + – + ∅ – + – + 194 bp Fold inuciferas 12 ##
100 bp 135 bp l 0
IL-13 – + – + – + – +
Nr5a2 Arg1
Nr5a2 wt Nr5a2 mt
Figure1|IL-13-mediatedLRH-1geneexpressionisdependentonSTAT6.(a)Nr5a2mRNAexpressioninthecolon,liver,peritonealmacrophages(MF),
B(LB)andT(LT)lymphocytesfromC57BL/6micedeterminedusingRT–PCR.InsetshowsinsituhybridizationofNr5a2mRNAinperitonealmacrophages
fromC57BL/6mice(scalebar,25mm).(b,c)Nr5a2mRNAexpressioninmacrophagesfromC57BL/6mice(b)andinhumanmacrophages(c)treatedwith
theindicatedcytokinesfor4h,determinedusingRT–PCR.Theresultswererepresentedinfoldinductionrelativetotheuntreatedcontrolorwild-type
littermate.(d,e)Nr5a2mRNAexpressioninmacrophagesfromC57BL/6micepretreatedwithAG490andstimulatedwithIL-13(d)andinmacrophages
fromStat6þ/þ andStat6(cid:2)/(cid:2) micestimulatedwithIL-13for4h(e),determinedusingRT–PCR.Theresultswererepresentedinfoldinductionrelativeto
theuntreatedcontrolorwild-typelittermate.(f)ImmunoblotanalysisofthenuclearexpressionofLRH-1andTBP(Tata-bindingprotein)inmacrophages
fromStat6þ/þ andStat6(cid:2)/(cid:2) micestimulatedwithIL-13for24h.(g,h)LuciferaseactivityinmacrophagesfromC57BL/6micetransfectedwithLRH-1
(LRH-1-luc)promoterconstructpretreatedwithAG490(g)orfromStat6þ/þ andStat6(cid:2)/(cid:2) mice(h),andtreatedwithIL-13for24h.Theresultswere
representedinfoldinductionrelativetotheuntreatedcontrolorwild-typelittermate.(i)SchematicpresentationofthefourputativeSTAT6response
elementsinthemouseNr5a2promoteridentifiedbyGenomatixalgorithmandassessmentofSTAT6recruitmenttosite1andtotheArg1promoter
determinedwiththeChIPanalysisusinggenomicDNAfromC57BL/6macrophagestreatedwithIL-13for4h.(j)Luciferaseactivityinmacrophagesfrom
Stat6þ/þ andStat6(cid:2)/(cid:2) micetransfectedwithLRH-1promoterconstructsandtreatedwithIL-13for18h.Theresultswererepresentedinfoldinduction
relativetotheuntreatedcontrol.Resultscorrespondtomean±s.e.m.oftriplicates.Dataarerepresentativeofthreeindependentexperiments.*Po0.05
**Po0.01comparedwiththerespectiveuntreatedcontrolandxPo0.05,xxPo0.01comparedwithIL-13-treatedwild-typelittermate.Pvalueswere
determinedusingBonferroni–Dunnettmethod.
Mutation of the STAT6-RE abolished the activity of the Nr5a2 were crossed with transgenic mice that express the Cre
reporter construct in response to IL-13 in Stat6þ/þ and recombinase under the control of the mouse phagocyte-selective
Stat6M(cid:2)/(cid:2) macrophages (Fig. 1j). These results demonstrate lysozyme promoter21,26. Compared with control (Lrh-1Mþ/þ)
that STAT6 directly controls the transcription of LRH-1 in macrophages, LRH-1 mRNA and protein levels were almost
response to IL-13. undetectable in macrophages derived from the myeloid cell-
specific LRH-1-deficient (Lrh-1M(cid:2)/(cid:2)) mice (Supplementary
LRH-1isinvolvedinIL-13-inducedmacrophageactivation.In Fig. 1a–c). Furthermore, the disruption of LRH-1 could not be
order to assess the role of LRH-1 in IL-13-induced alternative detectedinotherLRH-1-expressingtissues,suchastheliverand
macrophage differentiation, we generated mice in which the the colon (Supplementary Fig. 1d,e).
Nr5a2 gene was selectively disrupted in myeloid-derived cells. We then evaluated the expression of specific markers of
To generate these animals, mice carrying floxed Lrh-1 alleles classical and alternative activation in untreated or IL-13-treated
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Lrh-1Mþ/þ and Lrh-1M(cid:2)/(cid:2) macrophages during 4h. Overall, such as Nos2, Itgam (CD11b), Fcgr3, Fcgr1, Il1b and Il-6 still
Lrh-1M(cid:2)/(cid:2) macrophages displayed an upregulation of M1 remained highly expressed (Fig. 2a–c). Consistent with these
markers such as Nos2 (encoding the inducible nitric oxide findings, the induction of alternative activation gene markers
synthase)andtheFcg-receptorsFcgr3andFcgr1(encodingCD16 observed after 4h of IL-13 treatment was amplified after 24h of
and CD64 proteins, respectively), which was mirrored by a IL-13 treatment in Lrh-1Mþ/þ macrophages (Supplementary
downregulationofChi3l3(YM1),Mrc1(MR),Clec7a(Dectin-1), Fig.2a).Moreover,thedecreaseinalternativeactivationmarkers
Il1rn (IL-1ra) and Tgfb1 (transforming growth factor (TGF)-b1) in Lrh-1M(cid:2)/(cid:2) macrophages after 4h of IL-13 treatment was
alternative activation markers (Fig. 2a,b). This was accompanied sustained after 24h of stimulation (Supplementary Fig. 2a).
by an increase in the mRNA and protein levels of the Altogether, these data indicate that LRH-1 is required for
inflammatory cytokines TNFa, IL-1b and IL-6 (encoded by repression of pro-inflammatory state and for optimal induction
Tnfa, Il1b and Il-6 genes, respectively; Fig. 2a,c). Il12 pro- ofalternativemacrophageactivationbyIL-13.Thesefindingsare
inflammatory and Il10 anti-inflammatory cytokine mRNA levels consistent with the robust induction of Il10, Tgfb1, Il1rn, Mrc1,
remained unchanged in Lrh-1Mþ/þ and Lrh-1M(cid:2)/(cid:2) macro- Clec7a and Cd36 gene expression in Lrh-1Mþ/þ macrophages
phages (Fig. 2a). Furthermore, the induction of MR, Dectin-1, treated with the LRH-1 agonist DLPC (Supplementary Fig. 2b).
CD36, Arg1 (encoding the arginase 1), Chi3l3 and Il1rn
expression by IL-13 was strongly diminished in Lrh-1M(cid:2)/(cid:2)
macrophages (Fig. 2a,b). Consistent with reduced alternative LRH-1 activates 15-HETE secretion via the control of CYP1s.
activationmarkersinLrh-1M(cid:2)/(cid:2) macrophages,theM1markers ThenuclearreceptorPPARgisakeycomponentofthesignalling
Nos2 Arg1 Itgam Fcgr3 C IL-13
2 # 2 ** 2 # 2.5 # 104 104
*
1.5 # 1.5 1.5 2 # 103 8.62% 103 31.34%
* ## 1.5
0.15 * 0.15 0.15 * 0.15 Lrh-1M+/+ CD36110021 CD36110012
0 0 0 0 100 100
IL-13 – + – + – + – + – + – + – + – + 100 101 102 103 104 100 101 102 103 104
Dectin-1 Dectin-1
A expression level 43210 Fcg#r#1## 123450 *M*rc1#### 3210 C**lec7#a## 3210....35012555 *C*d36 ## LLrrhh--11MM+–//–+ Lrh-1M–/– CD36 111110000031420100 101 1029.15013%104 CD36111110000031420100 101 102 1150.130%104
NIL-13 – + – + – + – + – + – + – + – +
R Dectin-1 Dectin-1
m
elative 20 *C*hi3l3 1.5 *II1 rn 1.5 II-12 2 Tnf#α# 104 C 104 IL-13
R 11550 *#* 0.15 # # 0.15 01..155 * #* Lrh-1M+/+ FL-2111000321 14.42% FL-2 111000312 49.29%
##
0 0 0 0 100 100
IL-13 – + – + – + – + – + – + – + – + 100 101 102 103 104 100 101 102 103 104
MR MR
II1b II6 II10 Tgfb1
4 #### 2.5 ## 1.5 1.2 104 104
**
3 2 103 103
21 10..155 0.15 00..84 * # #* Lrh-1M–/– FL-2110021 14.11% FL-2 110021 27.41%
0 0 0 0 100 100
IL-13 – + – + – + – + – + – + – + – + 100 101 102 103 104 100 101 102 103 104
MR MR
IL-1β TNFα TGFβ1 Lrh-1M+/+
Cytokine release–1(pg ml) 213000000 ## #*# 1162840000 ## #*# 543210000000000 # Lrh-1M–/– Geo-meanfluorescence 963000 **MR ## 8642 D**ectin-1## 186420 *C*D36 ##
0 0 0 0 0 0
IL-13 – + – + – + – + – + – + IL-13 – + – + – + – + – + – +
Figure2|LRH-1isinvolvedinIL-13-inducedalternativeactivationofmacrophages.(a)GeneexpressionanalysisofmarkersofM1andM2polarization
inperitonealmacrophagesfromLrh-1Mþ/þ andLrh-1M(cid:2)/(cid:2) micetreatedwithIL-13for4h,determinedusingRT–PCR.Theresultswererepresented
infoldinductionrelativetotheuntreatedLrh-1Mþ/þ littermate.(b)Dot-plotrepresentingDectin-1,CD36andMRproteinexpressioninmacrophages
fromLrh-1Mþ/þ andLrh-1M(cid:2)/(cid:2) micetreatedwithIL-13for24h.Numbersindicatethe%ofpositivecells.Graphsrepresentgeomeanfluorescence
quantificationfortheindicatedproteins.(c)CytokineproductionofperitonealmacrophagesfromLrh-1Mþ/þ andLrh-1M(cid:2)/(cid:2) miceafterIL-13treatment
andC.albicanschallengefor8h(ratio:1macrophage:3yeasts),quantifiedbyenzyme-linkedimmunosorbentassay.Resultscorrespondtomean±s.e.m.of
triplicates.Dataarerepresentativeofthreeindependentexperiments.*Po0.05,**Po0.01comparedtotherespectiveuntreatedcontrolandxPo0.05,
xxPo0.01comparedwithLrh-1Mþ/þþIL-13.PvaluesweredeterminedusingtheBonferroni–Dunnettmethod.
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pathwaytriggeredbyIL-13anddirectlycontrolstheexpressionof PPARg promoter induction, further indicating that LRH-1 does
markers of alternative activation. To establish whether the notregulatethetranscriptionrateofPPARg.Next,weexamined
increase in alternative activation markers by IL-13 results from whether LRH-1 was required for PPARg activation by assessing
direct regulation of PPARg transcription by LRH-1, we first the impact of IL-13 on a heterologous PPARg reporter
evaluated Pparg mRNA levels in Lrh-1Mþ/þ and transfected in Lrh-1Mþ/þ and Lrh-1M(cid:2)/(cid:2) macrophages.
Lrh-1M(cid:2)/(cid:2) macrophages under basal conditions and after Remarkably, while in Lrh-1Mþ/þ macrophages IL-13 sig-
IL-13 exposure. The increased Pparg mRNA level by IL-13 in nificantlyinducedthePPREluciferasereporter,nosuchresponse
Lrh-1Mþ/þ macrophages was not affected in Lrh-1M(cid:2)/(cid:2) mac- could be observed in Lrh-1M(cid:2)/(cid:2) macrophages (Fig. 3d).
rophages (Fig. 3a). Moreover, in transient transfection studies, Conversely, co-transfection of the PPRE luciferase reporter with
absence of LRH-1 in Lrh-1M(cid:2)/(cid:2) macrophages (Fig. 3b) or an expression vector for LRH-1 robustly increased PPARg acti-
conversely ectopic expression of LRH-1 in wild-type macro- vation (Fig. 3e), suggesting that LRH-1 induces the activity of
phages (Fig. 3c) did not significantly affect IL-13-mediated PPARg.
Relative mRNAexpression level 10..51502 *Pparg * LLrrhh--11MM+–//–+ Luciferase activityfold induction3012 P*PARγ-luc * LLrrhh--11MM+–//–+ Luciferase activityfold induction012345 P*P*ARγ-luc * Luciferase activityfold induction01234 PPR**E-luc LL#rr#hh--11MM–+//–+ Luciferase activityfold induction 69100500 P*P*RE-lu#c# #*#*
IL-13 – + – + IL-13 – + – + IL-13 – + – + IL-13 – + – + IL-13 – + – +
Empty LRH-1 Empty LRH-1
Relative mRNAexpression level 01..0551 Ptgs2 001...4820 **Alox5 ** 012 Hpgds 012345 *A*lox15 #**# 0110....82640 *Cyp1a#1 #*# 11124680240 **Cyp1b#1LLrrhh#*--#11MM–+//–+ CYPA1cBtin1 Lrh-1M+/+Lrh-1M–/–Lrh-1M+/+ Lrh-1M–/–k45D35
IL-13 – + – + – + – + – + – + – + – + – + – + – + – + IL-13 – – + +
Alox15 Cyp1a1 Cyp1b1 Alox15 Cyp1a1 Cyp1b1
Relative mRNAexpression level 012 * ## 0123 ** # ## 012345 ** ## ## SSttaatt66+–//–+ Relative mRNAexpression level 0123 * ## 0123 ** # ## 01234 ** # LL#rrhh#--11MM+–//–+
IL-13 – + – + – + – + – + – + DLPC – + – + – + – + – + – +
Fold induction012345 15-H*E*TE production# LLrrhh--11MM+–//–+ 35 dpm/4.10 cells10115050000 [3H]*A*A mobilization** LLrrhh--11MM+–//–+ Fold induction 3012 ** 1#5#-HETE prodcution ** ##AAllooxx1155+–//–+
IL-13 – + – + IL-13 – + – + IL-13 – + – + – + – +
siRNA siRNA siRNA siRNA
C Cyp C Cyp
Figure3|LRH-1activatesCYP1A1-andCYP1B1-dependent15-HETEproduction.(a)PpargmRNAexpressioninmacrophagesfromLrh-1Mþ/þ and
Lrh-1M(cid:2)/(cid:2) micetreatedwithIL-13for4h,determinedusingRT–PCR.Theresultswererepresentedinfoldinductionrelativetotheuntreatedwild-type
littermate.(b)LuciferaseactivityinmacrophagesfromLrh-1Mþ/þ andLrh-1M(cid:2)/(cid:2) micetransfectedwithPPARg(PPARg-luc)promoterconstructand
treatedwithIL-13for4h.Theresultswererepresentedinfoldinductionrelativetotherespectivecontrol.(c)Luciferaseactivityinmacrophagesfrom
C57BL/6miceco-transfectedwithPPARg(PPARg-luc)promoterconstructinpresence(LRH-1)orabsence(empty)ofLRH-1(pCMX-LRH-1)andtreated
withIL-13for4h.Theresultswererepresentedinfoldinductionrelativetotheuntreatedcontrol(empty).(d)Luciferaseactivityinmacrophagesfrom
Lrh-1Mþ/þ andLrh-1M(cid:2)/(cid:2) micetransfectedwithaPPRE(PPRE-luc)constructtreatedwithIL-13for24h.Theresultswererepresentedinfoldinduction
relativetotherespectiveuntreatedcontrol.(e)LuciferaseactivityofmacrophagesfromC57BL/6macrophagesco-transfectedwithPPRE(PPRE-luc)
constructinpresence(LRH-1)orabsence(empty)ofLRH-1(pCMX-LRH-1),treatedwithIL-13for24h.Theresultswererepresentedinfoldinduction
relativetotherespectivecontrol.(f)GeneexpressionanalysisofarachidonicacidmetabolicenzymesinmacrophagesfromLrh-1Mþ/þ andLrh-1M(cid:2)/(cid:2)
micetreatedwithIL-13for4h,determinedusingRT–PCR.TheresultswererepresentedinfoldinductionrelativetountreatedLrh-1Mþ/þ.(g)Immunoblot
analysisofCyp1b1andActininmacrophagesfromLrh-1Mþ/þandLrh-1M(cid:2)/(cid:2) micestimulatedwithIL-13for24h.(h,i)GeneexpressionanalysisofAlox15,
Cyp1a1andCyp1b1inmacrophagesfromStat6þ/þ andStat6(cid:2)/(cid:2) micetreatedwithIL-13(h)andinmacrophagesfromLrh-1M(cid:2)/(cid:2) andLrh-1Mþ/þ
micestimulatedwithDLPCfor4h(i),determinedusingRT–PCR.Theresultswererepresentedinfoldinductionrelativetountreatedwild-typelittermate.
(j)15-HETEproductionbymacrophagesfromLrh-1M(cid:2)/(cid:2) andLrh-1Mþ/þ micestimulatedwithorwithoutIL-13quantifiedbyenzymeimmunoassay(EIA).
TheresultswererepresentedinfoldinductionrelativetountreatedLrh-1Mþ/þ.(k)[3H]AAmobilizationinmembranephospholipidsofmacrophagesfrom
Lrh-1M(cid:2)/(cid:2) andLrh-1Mþ/þ micestimulatedwithIL-13for2h.(l)15-HETEproductionbymacrophagesfromAlox15(cid:2)/(cid:2) andAlox15þ/þ micestimulated
withIL-13for24handsilencedornotforCyp1a1andCyp1b1(siRNACyp)measuredbyEIA.Theresultswererepresentedinfoldinductionrelativeto
respectiveuntreatedcontrol(siRNAC).Resultscorrespondtothemean±s.e.m.oftriplicates.Dataarerepresentativeofthreeindependentexperiments.
*Po0.05,**Po0.01comparedwiththerespectiveuntreatedcontrolandxPo0.05,xxPo0.01comparedwiththecorrespondingtreatedoruntreatedwild-
typelittermate.PvaluesweredeterminedusingBonferroni–Dunnettmethod.
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PPARg is activated by endogenous ligands derived from the promoters, while no conserved STAT6 REs (matrix similarity
metabolism of AA. The COX1/COX2 cyclooxygenases, 5 and o0.8) could be identified in these regulatory regions
12/15 lipoxygenases and CYP enzymes are considered to be (Supplementary Fig. 3a). Consistent with these findings, DLPC
criticalfortheconversionofAAintoendogenousPPARgligands. treatmentincreasedAlox15,Cyp1a1andCyp1b1geneexpression
To identify how LRH-1 may have an impact on PPARg in Lrh-1Mþ/þ macrophages, but not in Lrh-1M(cid:2)/(cid:2) macro-
activation, we next explored whether LRH-1 can coordinate phages(Fig.3i).ThesedataconfirmtheimportanceofLRH-1in
PPARgligandavailabilitythroughthecontroloftheexpressionof the regulation of Alox15, Cyp1a1 and Cyp1b1.
these enzymes. The mRNA levels of Ptgs2 (cyclooxygenase 2), Finally,toassesswhethertheseeffectsongeneexpressionalso
Alox5 (5 lipoxygenase) and Hpgds (prostaglandin-D synthase) translateintochangesinendogenousligandavailability,15-HETE
after IL-13 stimulation were not differentially expressed in production was assessed. Interestingly, while IL-13 exposure
Lrh-1Mþ/þ and Lrh-1M(cid:2)/(cid:2) macrophages (Fig. 3f). However, robustly enhanced 15-HETE levels in Lrh-1Mþ/þ macrophages,
IL-13robustlyinducedAlox15(12/15lipoxygenase),Cyp1a1and this effect was completely lost in Lrh-1M(cid:2)/(cid:2) macrophages
Cyp1b1 gene expression in Lrh-1Mþ/þ macrophages, while this (Fig.3j).ThesefindingsindicatethatLRH-1iscriticallyrequired
induction was blunted in Lrh-1M(cid:2)/(cid:2) macrophages. Moreover, for IL-13-induced 15-HETE production in macrophages.
Cyp1b1 protein levels were only induced in Lrh-1Mþ/þ Importantly, IL-13-induced mobilization of AA was similar in
macrophages on IL-13 exposure, but not in Lrh-1M(cid:2)/(cid:2) Lrh-1Mþ/þ and Lrh-1M(cid:2)/(cid:2) macrophages (Fig. 3k), indicating
macrophages(Fig.3g).UnlikeCyp1a1andCyp1b1mRNAlevels, that the generation of 15-HETE metabolites through LRH-1 is
which were unresponsive to the IL-13 treatment in Lrh-1M(cid:2)/(cid:2) dependent on AA metabolism.
macrophages, Alox15 expression was still moderately induced To further dissect how LRH-1 promotes the production of
(Fig. 3f), indicating that Alox15 is only partially controlled by 15-HETEsinresponsetoIL-13,weassessed15-HETEproduction
LRH-1. in Alox15-deficient macrophages on Cyp1a1 and Cyp1b1 short
Consistent with these findings, a strong decrease in Alox15, interfering RNA (siRNA)-mediated silencing (Fig. 3l and
Cyp1a1 and Cyp1b1 expression could be observed in both Supplementary Fig. 3b). Interestingly, the increased 15-HETE
untreated and IL-13-treated Stat6(cid:2)/(cid:2) macrophages (Fig. 3h), production by IL-13 was still conserved in Alox15(cid:2)/(cid:2) macro-
furthersupportingtheimportanceofSTAT6intheregulationof phages.Furthermore,thesimultaneousgenesilencingforCyp1a1
these genes. and Cyp1b1 in both Alox15 þ/þ and Alox15(cid:2)/(cid:2) macrophages
To further explore whether STAT6 controls the expression of abolished this induction (Fig. 3l). Altogether, these data indicate
Alox15, Cyp1a1 and Cyp1b1 directly or indirectly through the that LRH-1 drives the generation of 15-HETE metabolites
inductionofLRH-1,weperformedaninsilicoanalysisofAlox15, through its impact on CYP1 gene expression.
Cyp1a1 and Cyp1b1 promoters (Supplementary Fig. 3a). This To define whether Cyp1a1 and Cyp1b1 are direct transcrip-
analysis revealed one putative LRH-1 and two putative STAT6- tional targets of LRH-1, transfection assays in Lrh-1Mþ/þ and
REinthe Alox15 promoter,withmorethan95%of similarity to Lrh-1M(cid:2)/(cid:2) macrophages were performed using a luciferase
theconsensusREs.ScanningoftheCyp1a1andCyp1b1promoter reporter containing ±1.2kb of the promoter of the Cyp1a1 and
sequencesindicatedthepresenceofconservedLRH-1REsinboth Cyp1b1 genes. IL-13 exposure of Lrh-1Mþ/þ macrophages
Cyp1a1 mt1 Cyp1a1 mt2
14 –1165 Site 1 –1150–272 Site 2 –258 Cyp1a1 wt Cyp1a1 40 Cyp1a1 × 38 Lrh-1M+/+
Luciferase activityfold induction 11864220 * ## ** LL#rrhh#--11MM+–//–+ 12S3000it000e bbb1pppLrh-1+/I+P– /L–R+H/+-–1/–+/+I–n/–p+u/+t–/–+/+I–P/– Ig+/G+–/– 192 bp Relative binding 321000 × 10 × 1 × 0.L7rh-1M–/–
0 IL-13– – + + – – + + – – + + 0
IL-13 – + – + – + – + – + – + IL-13 – – + +
Cyp1a1 wt Cyp1a1 mt1 Cyp1a1 mt2
Cyp1b1mt
uciferase activityfold induction108642 ** –742 ## Site 1 –7C28yp1b1wt LLrrhh--11MM+–//–+ C321y000p0001 bbbbpp1pLrh-1+/I+P– L/–R+/H+–-/1– +/+I–n/–pu+/t+–/–+/+IP–/– Ig+/G+–/–150 bp Relative binding 321456000000 ×C y2p.51b×1 1 × 56× 0.8LLrrhh--11MM+–//–+
L 0 IL-13– – + + – – + + – – + + 0
IL-13 – + – + – + – + IL-13 – – + +
Cyp1b1 wt Cyp1b1 mt
Figure4|LRH-1controlsthetranscriptionofCyp1a1andCyp1b1genesinresponsetoIL-13.(a–c)LuciferaseactivityinmacrophagesfromLrh-1Mþ/þ
andLrh-1M(cid:2)/(cid:2) micetransfectedwithCyp1a1promoterconstructs(a)orCyp1b1promoterconstructs(c)andtreatedwithIL-13for18h.Theresultswere
representedinfoldinductionrelativetotheuntreatedcontrol.(b,d)AssessmentofLRH-1recruitmenttosite1totheCyp1a1promoter(b)orCyp1b1
promoter(d)determinedwiththeChIPanalysisusinggenomicDNAfromLrh-1Mþ/þ andLrh-1M(cid:2)/(cid:2) macrophagestreatedwithIL-13for18h.
Resultscorrespondtomean±s.e.m.oftriplicates.Dataarerepresentativeofthreeindependentexperiments.*Po0.05,**Po0.01comparedwiththe
respectiveuntreatedcontrolandxPo0.05,xxPo0.01comparedwiththecorrespondingtreatedwild-typelittermate.Pvaluesweredeterminedusing
theBonferroni–Dunnettmethod.
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NATURECOMMUNICATIONS|DOI:10.1038/ncomms7801
resulted in an eightfold increase in reporter activity of both macrophages (Fig. 5e). The lack of IL-13-augmented induction of
Cyp1a1 and Cyp1b promoters (Fig. 4a,c). Interestingly, genetic alternative markers was associated with a failure of Stat6(cid:2)/(cid:2)
deletion of LRH-1 abolished this response, demonstrating that macrophages to produce 15-HETE in response to IL-13 (Fig. 5f).
Cyp1a1 and Cyp1b1 promoters are directly activated by LRH-1. Interestingly, the addition of exogenous 15-HETE restored the
To identify the critical LRH-1 REs in the Cyp1a1 and Cyp1b1 induction of alternative polarization markers in Stat6(cid:2)/(cid:2)
promoters,wemutagenizedtheputativeREthatwerefoundbyin macrophages and not in PpargM(cid:2)/(cid:2) macrophages (Fig. 5e and
silico analysis (Supplementary Fig. 3a), and their response to SupplementaryFig.3c).ThesedatasuggestthatSTAT6isrequired
LRH-1 on IL-13 exposure was compared (Fig. 4a–c). For the for induction of macrophage-alternative activation markers and
Cyp1a1 promoter, mutation of the first LRH-1 RE (site 1) furthersupporttheexistenceofaPPARg-dependentmechanismin
abolished the activity of the reporter construct in response to theregulation of these genes.
IL-13, whereas mutation of site 2 was still responsive in Moreover,inductionofArg1,Retnla(Fizz1)andChi3l3(YM1)
Lrh-1Mþ/þ macrophages (Fig. 4a). Furthermore, whole inhibi- in response to IL-13 was slightly decreased in PpargM(cid:2)/(cid:2)
tion of mutated reporter construct activities in Lrh-1M(cid:2)/(cid:2) macrophages, whereas the induction of Mrc1 (MR), Clec7a and
macrophages established that site 1 is the principal site CD36 was completely abrogated in PpargM(cid:2)/(cid:2) macrophages
transmittingthe effect of LRH-1onthe Cyp1a1promoter.Thus, (Fig.5g).Theseresultsindicatetheexistenceofdistinctregulatory
thisresultidentifiedspecificrecruitmentofLRH-1tosite1,which mechanisms involving either STAT6 with a modest contribution
is most distal to the transcription initiation site in the Cyp1a1 of PPARg or predominantly controlled by the LRH-1/PPARg
promoter. axis. In line, the overexpression of Mrc1, Clec7a and Cd36 after
FortheCyp1b1promoter,IL-13treatmentfailedtoincreasethe treatment with DLPC in PpargMþ/þ macrophages was not
activity of the mutated Cyp1b1 reporter in both Lrh-1Mþ/þ or detectedinPpargM(cid:2)/(cid:2) macrophages(Fig.5h),clearlyestablish-
Lrh-1M(cid:2)/(cid:2) macrophages (Fig. 4c), indicating that LRH-1 binds ing that LRH-1 acts upstream from PPARg in the signalling
and activates the Cyp1b1 promoter through a unique sequence cascade leading to the PPARg-dependent gene expression.
between (cid:2)742 and (cid:2)728bp upstream of the transcription
initiation site of the gene. Finally, ChIP assays were performed.
IL-13 enhanced the recruitment of LRH-1 on both Cyp1a1 and IL-13-inducedfungicidalpropertiesofmacrophagesviaLRH-1.
Cyp1b1sitesinLrh-1Mþ/þ macrophages,butnotinLrh-1M(cid:2)/(cid:2) Previousworkfromourlaboratoryestablishedtheimportanceof
macrophages (Fig. 4b–d). Altogether, these results demonstrate PPARg in the fungicidal functions of alternatively activated
that LRH-1 directly binds Cyp1a1 and Cyp1b1 promoters and macrophages27.Onthebasisofthecurrentfindingssuggestinga
hence controls the transcription of Cyp1a1 and Cyp1b1 genes in role for LRH-1 in PPARg-mediated alternative polarization
response to IL-13. following IL-13 stimulation, we next investigated whether
deletion of LRH-1 in macrophages could have an impact on the
outcome of Candida albicans infection. The severe systemic
LRH-1/CYP1-dependent 15-HETE release induces PPARc
infectionofmicewithC.albicansresultedinasignificantlylower
activation. To further determine whether the generation of
survival rate of Lrh-1M(cid:2)/(cid:2) mice compared with Lrh-1Mþ/þ
15-HETE metabolites through LRH-1 are involved in PPARg
mice (Po0.001; Fig. 6a), supporting a role for LRH-1 in
activation,weassessedwhethersupplementationof15-HETEcan
antifungal defence. To further explore the exact function of
rescuethelossofPPARgactivationinLrh-1M(cid:2)/(cid:2) macrophages.
LRH-1 in the pathophysiology of fungal infection, we evaluated
In contrast to IL-13, which could not induce PPARg activation
the fungal burden in the intestinal tract and the macrophage
in Lrh-1M(cid:2)/(cid:2) macrophages, addition of exogenous 15-HETE
microbicidal functions in a murine experimental model of
efficiently restored the induction of both a PPRE luciferase
gastrointestinal candidiasis. Lrh-1M(cid:2)/(cid:2) mice infected with
reporter(Fig.5a)andofPPARgtargetgenessuchasMrc1,Clec7a
C. albicans had more severe gastrointestinal infection than their
and Cd36 (Fig. 5b) in Lrh-1M(cid:2)/(cid:2) macrophages, indicating that
wild-type littermates and showed worsened fungal burden in
the PPARg activation through LRH-1 is critically dependent on
the caecum (Fig. 6b). Remarkably, IL-13, 15-HETE, as well as
15-HETE production.
DLPC, diminished C. albicans gastrointestinal colonization in
To confirm that 15-HETE production through the LRH-1/
Lrh-1Mþ/þ mice.However,theseeffectswerelostinLrh-1M(cid:2)/(cid:2)
CYP1 axis induces PPARg activation, we evaluated PPARg
mice treated with IL-13 or DLPC, but not when the PPARg
activation in macrophages silenced for Cyp1a1 and Cyp1b1.
ligand, 15-HETE, was administered to the animals (Fig. 6b).
Interestingly,PPARgactivityasdeterminedbytheinductionofa
ToinvestigatewhetherLRH-1inmacrophageshasanyrelevant
PPRE luciferase reporter (Fig. 5c) and the induction of PPARg
microbicidalphenotype,weevaluatedthecapacityofLrh-1Mþ/þ
target genes (Fig. 5d) by IL-13 were totally inhibited in
and Lrh-1M(cid:2)/(cid:2) macrophages to kill yeasts in vitro. Compared
macrophages deficient for Cyp1a1 and Cyp1b1. Moreover, the
with Lrh-1Mþ/þ macrophages, Lrh-1M(cid:2)/(cid:2) macrophages
induction of a PPRE luciferase reporter (Fig. 5c) and of PPARg
showedadefectintheirabilitytokillC.albicans,demonstrating
targetgenes(Fig. 5d)wasstillsignificantlyenhancedbyIL-13in
the contribution of LRH-1 in macrophage-intrinsic antifungal
Alox15(cid:2)/(cid:2) macrophages,showingthatthe12/15lipoxygenaseis
activity (Fig. 6c). Consistent with our observation, Lrh-1M(cid:2)/(cid:2)
not required for PPARg activation mediated by LRH-1. These
macrophages were less efficient in engulfing C. albicans and
data are in support of a critical role of CYP1A1 and CYP1B1 in
producing reactive oxygen species (ROS) after fungal challenge
LRH-1-mediated PPARg activation through 15-HETE synthesis.
(Fig. 6d,e). Moreover, the defect of Lrh-1M(cid:2)/(cid:2) macrophages to
exert their antifungal activity was correlated with lower MR and
IL-13activationofmacrophagesrequiresSTAT6/LRH-1/PPARc. Dectin-1proteinlevelsafterC.albicanschallenge(Supplementary
To determine whether STAT6 controls both directly the tran- Fig. 3d). As expected, treatment with IL-13 of Lrh-1Mþ/þ
scription of markers of IL-13-mediated alternative activation and macrophages increased the killing and the phagocytosis of
indirectly through the activation of the LRH-1/PPARg axis, we C. albicans and also ROS production in response to C. albicans.
studied the mRNA level of alternative activation markers in These inductions were abrogated in Lrh-1M(cid:2)/(cid:2) macrophages,
STAT6-deficient macrophages. IL-13-augmented induction of underscoring the importance of LRH-1 in these fungicidal
Arg1, Chi3l3 (YM1), Retnla (Fizz1), MR, Clec7a and CD36 was functions (Fig. 6c–e). Similar effects were obtained when
detected in Stat6þ/þ macrophages but not in Stat6(cid:2)/(cid:2) macrophages were stimulated with DLPC (Fig. 6c–e).
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PPRE-luc Mrc1 Clec7a Cd36
Luciferase activityfold induction43210 ** ** ## ** LLrrhh--11MM+–//–+ Relative mRNAexpression level453210 ** * ## ## * 3210 ** * ## * 2310....55553210 ** * #LL#rrhh--11MM*+–//–+
IL-13 – + – – + – IL-13 – + – – + – – + – – + – – + – – + –
15-HETE – – + – – + 15-HETE – – + – – + – – + – – + – – + – – +
Luciferase activityfold induction43210 PPRE*-*luc ## ** AAlLoox#x11#55+–/+/– Relative mRNAexpression level0123 Mrc1** ## ** ## 10..10552 Clec7*a* # * ## 210...215550 Cd36** ## * AAlLooxx1155+#–/+/#–
IL-13 – + – + – + – + IL-13 – + – + – + – + – + – + – + – + – + – + – + – +
siRNA siRNA siRNA siRNA siRNA siRNA siRNA siRNA siRNA siRNA siRNA siRNA siRNA siRNA siRNA siRNA
c Cyp C Cyp c Cyp C Cyp c Cyp C Cyp C Cyp C Cyp
Arg1 Retnla Chi3l3 Mrc1 Clec7a Cd36 15-HETE production
Relative mRNAexpression level1086420 ** ** ####** 012345 ** ** # #*# ** 11102486420 ** * ## ** 1864200 ** ** # ##** 6012345 ** * ## * 501234 ** ** SS#*#ttaatt66**+–//+– Fold induction0312 ** SS#tt#aatt66+–//+–
IL-13 – + – – + – – + – – + – – + – – + – – + – – + – – + – – + – – + – – + – IL13 – + – +
HETE – – + – – + – – + – – + – – + – – + – – + – – + – – + – – + – – + – – +
Arg1 Retnla Chi3l3 Mrc1 Clec7a Cd36 Mrc1 Clec7a Cd36
Relative mRNAexpression level012345 * #* 12332155500005 ** #* 123215500005 ** #*# 01234 ** # ## 012...555021 ** # ## 012345 ** PP#ppaarr#gg#MM+–//–+ Relative mRNAexpression level 0123456789 ** # ## 0123 ** ## ## 01234 ** #PP#ppaa#rrgg#MM+–//–+
IL-13 – + – + – + – + – + – + – + – + – + – + – + – + DLPC – + – + – + – + – + – +
Figure5|STAT6/LRH-1/PPARcsignalingisrequiredforIL-13-mediatedalternativeactivationofmacrophages.(a)Luciferaseactivityinperitoneal
macrophagesfromLrh-1Mþ/þ andLrh-1M(cid:2)/(cid:2) micetransfectedwithaPPRE(PPRE-luc)constructandtreatedwithIL-13or15-HETEfor24h.
(b)GeneexpressionanalysisofMrc1,Clec7aandCd36inmacrophagesfromLrh-1Mþ/þ andLrh-1M(cid:2)/(cid:2) micetreatedwithIL-13or15-HETEfor4h,
determinedbyRT–PCR.(c)LuciferaseactivityofmacrophagesfromALox15þ/þ andALox15(cid:2)/(cid:2) micetransfectedwithaPPRE(PPRE-luc)constructand
siRNAtargetingCyp1a1andCyp1b1(siRNACyp)andtreatedwithIL-13for24h.(d)GeneexpressionanalysisofMrc1,Clec7aandCd36inmacrophages
fromALox15þ/þ andALox15(cid:2)/(cid:2) micetransfectedwithsiRNAtargetingCyp1a1andCyp1b1(siRNACyp)treatedwithIL-13for4handdeterminedby
RT-PCR.(e,g)GeneexpressionanalysisofArg1(arginase1),Retnla(Fizz1),Chi3l3(YM1),Mrc1,Clec7aandCd36inmacrophagesfromStat6þ/þ and
Stat6(cid:2)/(cid:2) mice(e)orfromPpargMþ/þ andPpargM(cid:2)/(cid:2) mice(g)treatedwithIL-13or15-HETE(e)for24h,determinedbyRT-PCR.(f)15-HETE
productionbymacrophagesfromStat6(cid:2)/(cid:2) andStat6þ/þ micestimulatedwithIL-13for24hmeasuredbyEIA.(h)GeneexpressionanalysisofMrc1,
Clec7aandCd36inmacrophagesfromPpargMþ/þ andPpargM(cid:2)/(cid:2) treatedwithDLPCfor4h,determinedbyRT-PCR.Resultswererepresentedinfold
inductioncomparedtotherespectiveuntreatedcontrolorwild-typelittermateandcorrespondtomean±s.e.m.oftriplicates.Dataarerepresentativeof
threeindependentexperiments.*Po0.05,**Po0.01comparedtotherespectivefloxedornotuntreatedcontrolandxPo0.05,xxPo0.01comparedtothe
correspondinguntreatedortreatedwild-typelittermateorsiRNAcontrol.PvaluesweredeterminedusingBonferroni–Dunnettmethod.
Interestingly, treatment with 15-HETE increased the fungicidal previously demonstrated that IL-13, via the cPLA signalling
2
functions in both Lrh-1Mþ/þ and Lrh-1M(cid:2)/(cid:2) macrophages pathway, induced AA mobilization associated with the nuclear
(Fig.6c–e).Moreover,treatmentwithIL-13,DLPCand15-HETE localization of 15d-PGJ2, an endogenous PPARg ligand5. Once
of PpargM(cid:2)/(cid:2) macrophages did not increase the killing of activated, PPARg induces the transcription of Dectin-1, MR
C. albicans (Fig. 6f), corroborating our findings that PPARg is and CD36, three genes characteristic of the alternative
downstream from LRH-1 in the signalling pathway triggered by activation5,30,31. Therefore, the processes leading to PPARg
IL-13, leading to macrophage fungicidal activities. activation, such as AA release and its subsequent metabolic
TounequivocallyestablishthattheLRH-1/CYP1/HETEaxisis conversion,couldbeimportantaspectsofalternativepolarization
involvedin macrophage-intrinsic antifungal activity of IL-13, we because they are limiting factors for PPARg ligand synthesis.
evaluated the ability of macrophages silenced for Cyp1a1 and AA can be metabolized by the COX1/COX2 cyclooxygenases
Cyp1b1 (Cyp1) to kill C. albicans. Interestingly, the increase in toPGH2,whichinturnistransformedbythePGDsynthaseinto
C. albicans killing by IL-13 and DLPC was inhibited by the 15d-PGJ2 (refs 32,33). AA can also be directly metabolized to
simultaneousgenesilencingforCyp1a1andCyp1b1(Cyp1),but 12-andHETEs,otherendogenousPPARgligands,through12/15
not after 15-HETE stimulation (Fig. 6g). Taken together, these lipoxygenases34. A third pathway of AA metabolism leading to
data provide in vivo evidence that LRH-1 is involved in the endogenous PPARg ligand production is associated with its
PPARg-dependentantifungalfunctionselicitedbyIL-13through conversion by the enzymes of the CYP family35–37. The CYP
CYP1-induced 15-HETE production. enzymesgeneratetwobiologicalandactiveclassesofeicosanoids,
the epoxy (EETs) and hydroxy (HETEs) derivatives10,11. The
Discussion CYP1 family is mainly involved in the formation of mid-chain
The nuclear receptor PPARg is essential for IL-13-induced HETEs, such as 12- and 15-HETEs, through CYP1A1 and
alternative differentiation of macrophages6,28,29. We have CYP1B1 (refs 12,13).
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100
80 LLrrhh--11MM+–//–+ Cecum Lrh-1M+/+ Killing Lrh-1M+/+
Survival (%) 6420000 ** 6Yeast number (10)per mg of tissue 110000......01428642 # * # * # *Lrh*-1M–/– Killing (%) 142860000000 # ** ## ** ##Lrh-1*M–/*–*
0 2 4 6 8 10 12 14 16 18 20 C IL-13 DLPC 15-HETE C IL-13 DLPC15-HETE
Days post infection
Phagocytosis ROS production Killing Killing
Lrh-1M+/+ Lrh-1M+/+ PpargM+/+ siRNA C
Fold induction 1100....6284 # ** ## ** ##Lrh-*1M*–/– Fold induction 21 # ** ## ** ##L*rh*-1M*–/– Killing (%) 24680000 # * ## *P#p#argM*–/–# Killing (%) 82460000 ** # ** #s#iRNA* Cy*p1
0 0 0 0
C IL-13 DLPC15-HETE C IL-13 DLPC15-HETE C IL-13 DLPC15-HETE C IL-13 DLPC15-HETE
Figure6|IL-13-inducedantifungalpropertiesofmacrophagesrequireLRH-1.(a)SurvivalofLrh-1M(cid:2)/(cid:2) andLrh-1Mþ/þ micetoanintraperitoneal
injectionofC.albicans(1.108yeastspermouse,n¼32pergroup).Survival(%)wasassessedtwicedaily.*Po0.001comparedwithLrh-1Mþ/þ miceusing
log-ranktest.(b)Lrh-1Mþ/þ andLrh-1M(cid:2)/(cid:2) micewereinfectedwithC.albicans,andtreatedi.p.without(C)orwithIL-13,DLPCor15-HETE.C.albicans
gastrointestinalcolonizationinthecaecumwasdeterminedonday7usingRT–PCR.Dataarerepresentedasmean±s.e.m.*Po0.05comparedwiththe
respectiveuntreatedcontrolandxPo0.05comparedwiththecorrespondinguntreatedortreatedLrh-1Mþ/þ.Thedataarerepresentativeofatleasttwo
independentexperiments(n¼10pergroup).(c)KillingassayofLrh-1Mþ/þ andLrh-1M(cid:2)/(cid:2) macrophagesincubatedwithC.albicans.(d,e)Phagocytosis
(d)andROSinduction(e)ofC.albicansweremeasuredinmacrophagesfromLrh-1M(cid:2)/(cid:2) andLrh-1Mþ/þ mice.Dataareexpressedasfoldinduction
relativetothefluorescence(c)orchemiluminescence(d)observedforuntreatedLrh-1Mþ/þ.(f)KillingassayofPpargMþ/þ andPpargM(cid:2)/(cid:2)
macrophagesincubatedwithC.albicans.(g)KillingassayofmacrophagessilencedforCyp1a1andCyp1b1incubatedwithC.albicans.*Po0.05,**Po0.01
comparedwiththerespectiveuntreatedcontrolandxPo0.05xxPo0.01comparedwiththecorrespondingtreatedwild-typelittermateorsiRNAcontrol.
PvaluesweredeterminedusingBonferroni–Dunnettmethod.Resultscorrespondtomean±s.e.m.oftriplicatesandarerepresentativeofatleastthree
independentexperiments.Forindicatedmeasurements,treatmentswithIL-13,15-HETEandDLPCwereperformed24hbeforethechallengewith
C.albicans.
HerewereportthatthenuclearreceptorLRH-1isexpressedin Despite the growing knowledge with regard to the biological
macrophages and in response to IL-13 directly binds CYP1A1 functionofLRH-1,littleisknownabouthowLRH-1iscontrolled
andCYP1B1promoterstopositivelyregulatetheirtranscription. at the transcriptional level. We identified STAT6 as a transcrip-
Moreover, 15-HETE production following IL-13 stimulation is tionalregulatorofLRH-1.Thiswasevidencedbytheinductionof
impaired in macrophages deficient for LRH-1 and not in LRH-1 promoter activity by binding of STAT6 to its RE in the
macrophages lacking 12/15 lipoxygenase, indicating that LRH-1 LRH-1 promoter and by the decrease in LRH-1 mRNA and
drivesthegenerationof15-HETEmetabolitesthroughitsimpact proteinlevelsinmacrophageslackingSTAT6.Onthebasisofthe
onCYP1geneexpression.Consistently,ourfindingsshowingthat established role of STAT6 in PPARg activation and macrophage
the concurrent gene silencing of Cyp1a1 and Cyp1b1 in polarization41, these findings identify LRH-1 as a critical
macrophages abolishes the generation of 15-HETE, provide component in the signalling cascades that drive PPARg-
evidence that its production through the LRH-1/CYP1s axis is mediated alternative macrophage activation. This was further
crucialinPPARgactivation.Thisiscorroboratedbythefindings highlighted by the fact that macrophages lacking LRH-1 present
that PPARg activation on IL-13 stimulation is lost in macro- an increase in pro-inflammatory cytokines and the simultaneous
phages silenced simultaneously for CYP1A1/CYP1B1 and expression of other M1 markers. The involvement of LRH-1 in
restored by the addition of exogenous 15-HETE in macrophages anti-inflammatory responses was supported by the robust
lacking LRH-1. Consistent with these observations, treatment of reduction of LRH-1 gene expression in response to Th1
macrophages with the LRH-1 agonist, DLPC, increased the cytokines and conversely by the upregulation by Th2 cytokines.
expression of CD36, MR and Dectin-1 PPARg target genes in Interestingly,LRH-1wasalsoinducedinhumanmacrophagesin
wild-type macrophages but not in macrophages lacking PPARg. responsetotheTh2cytokineIL-13viaamechanismthatismost
Altogether,theseresultsestablishthatPPARgactivationbyIL-13 likely also STAT6-dependent, given the presence of several
is dependent on the LRH-1/CYP1/15-HETE pathway. Another conserved STAT6 REs in the human LRH-1 promoter
endogenous activator to consider in PPARg activation is 15d- (Supplementary Fig. 3a). Our findings may further explain why
PGJ2. Although we have previously shown that IL-13 generates during Crohn’s disease, characterized by a Th1 cytokine profile,
15d-PGJ2 production and its nuclear localization in macro- mRNA expression levels of LRH-1 are lower than in ulcerative
phages5,theresultsinthisstudysuggestthatitisnotsufficientto colitis, characterized by a Th2 immune response21. Consistent
activate PPARg. This is supported by previous reports showing with the anti-inflammatory role of LRH-1, IL-13-induced
that 15d-PGJ2 concentration required to stimulate PPARg is in alternative activation was impaired in macrophages lacking
the mM range, in contrast to other prostaglandins that are LRH-1. Indeed, on IL-13 treatment, the induction of several
normally active at low nM concentrations38,39. Thus, the levels signature genes of alternative activation, including Arginase 1,
generated in vivo are not sufficient to be compatible with a role YM1, IL-1 receptor antagonist (IL-1ra), MR, Dectin-1 and CD36,
for this metabolite as an endogenous PPARg ligand38,40. wassignificantlyimpairedinmacrophageslackingLRH-1.Thisis
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ARTICLE
NATURECOMMUNICATIONS|DOI:10.1038/ncomms7801
in agreement with reports showing that LRH-1 controls the IL-13
expression of anti-inflammatory IL-1ra and the scavenger ROS
Fungicidal
receptor class B type I, two markers specific of alternatively response
activated macrophages22,23,42. STAT-6 cPLA2Phagocytosis
AA
In addition to the key role of LRH-1 in the acquisition of
Cyp1a1
alternative activation of macrophages, this study also provides Dectin-1
Cyp1b1
mechanistic insight into the hierarchy between STAT6, LRH-1
andPPARgtoachievethisphenotype.Ourfindingsshowingthat ↑ LRH-1 15-HETE C. albicans
mlosascroofpihnadguecstcioannboefraelstetornreadtivbeyaecxtoigveantioounsm15a-rHkeErTsEinsuSptapto6r(cid:2)tt/h(cid:2)e Gene transcription
notion that STAT6 is required for macrophage-alternative PPARγ Induction of alternative Mannose receptor
activation programme
activation through PPARg-dependent mechanism. Moreover,
the use of PpargM(cid:2)/(cid:2) macrophages provides evidence for the
MΦ
existenceofdistinctmechanismsinthetranscriptionalregulation
of genes characteristics of alternative activation. Our results
demonstrate that the transcriptional regulation of Arginase 1,
Fizz 1 and YM1 involves directly STAT6 with a modest
contribution of PPARg and that Dectin-1, MR and CD36 are Figure7|SchematicillustrationoftheroleofLRH-1inIL-13-alternative
regulated indirectly by STAT6 through the LRH-1/PPARg axis. activationprogramofmacrophagesandinassociatedfungicidal
These observations are not only consistent with the requirement activities.ThealternativepolarizationofmacrophagesbyIL-13is
ofSTAT6toinducethemajorityofPPARgtargetgenes41butalso dependentontheincreaseofLRH-1expressionthroughSTAT-6which
with the identification of PPARg as a positive regulator of controlstheexpressionofCyp1a1andCyp1b1enzymesleadingtothe
alternative activation6. generationof15-HETEPPARgligand.
Consistent with the involvement of the LRH-1/PPARg path-
way in inducing MR and Dectin-1 expression during IL-13- werepurchasedfromJacksonLaboratories.PpargM(cid:2)/(cid:2) micedeletedforPparg
mediated alternative activation, loss of LRH-1 and PPARg in specificallyinmacrophageshavebeendescribedearlier30,43.Nr5a2(encoding
macrophages also severely compromised their capacity to kill, to LRH-1)macrophagespecificknockoutmice(referredasLrh-1M(cid:2)/(cid:2) mice)were
engulf C. albicans and to produce ROS. This is in line with the obtainedbycrossingmicecarryingfloxedLrh-1alleleswithtransgenicmice
expressingtheCrerecombinaseunderthecontrolofthemousephagocyte-selective
fact that LRH-1 is upstream from PPARg in the signalling lysozymepromoter21,26.ForLrh-1M(cid:2)/(cid:2) andPpargM(cid:2)/(cid:2) mice,the
pathwayleadingtotheinductionofMRandDectin-1,twoC-type correspondingfloxedlittermateswereusedascontrolsthroughoutallthe
lectin receptors strongly involved in the antifungal functions of experiments.CorrespondinglittermateswereusedascontrolsforStat6(cid:2)/(cid:2)
macrophages against C. albicans5,27,31. LRH-1 deficiency in andALox15(cid:2)/(cid:2) mice.
Fortheinvivoexperiments,agastrointestinalinfectionwiththeC.albicans
myeloid cells also rendered the mice highly susceptible to
strainwasestablishedbygavagewith50(cid:3)106C.albicanspermouse(n¼10per
gastrointestinal and systemic C. albicans infection, highlighting group).Miceweretreatedornotintraperitoneally(i.p.)withIL-13(Clinisciences),
LRH-1 of myeloid lineage as a key effector of host fungicidal DLPC(Sigma)or15-HETE(Cayman).ForIL-13treatment,injectionsof4mgper
functions. Although we have not characterized the role of mousewereperformed1daybeforeand3daysaftertheinfectionwithC.albicans
(twoinjections).ForDLPC(300mgper10gofmouse)and15-HETE(28mgper
neutrophils in this infectious context, our in vitro and in vivo
10gofmouse),i.p.injectionswererealized1daybeforethedayoftheinfection
results identify LRH-1 as a nuclear receptor indispensable for
withC.albicansandthenevery2days(fiveinjections).Controlgroupsreceived
alternative activation of macrophages and for its associated salinesolutiononlywithDMSO.After6daysofinfection,thececawereremoved
antifungal functions. asepticallyfortheexperiments.
In conclusion, we have shown that loss of LRH-1 in ForC.albicanssystemicinfection,yeastswereadministeredi.p.
(100(cid:3)106yeastspermouse).Survivalstudieswereconductedusing32mice
macrophages prevents IL-13-induced alternative activation of
pergroupandwererepeatedtwice.
macrophages, demonstrating the pivotal role of LRH-1 in the
differentiationofmacrophagestowardsananti-inflammatoryand
Humanmacrophages. Monocyteswereobtainedfromhealthyblooddonors
antifungal phenotype. In response to IL-13, LRH-1 expression is
(EtablissementFranc¸aisduSang,EFSToulouse).Writteninformedconsentswere
increased in macrophages through STAT6 and controls the obtainedfromthedonorsunderEFScontractno.21/PVNT/TOU/UPS04/2010–
expressionofCYP1A1andCYP1B1enzymes,whichcatalysesthe 0025.FollowingarticlesL1243-4andR1243-61oftheFrenchPublicHealthCode,
generation of 15-HETE PPARg ligand. Altogether, these results thecontractwasapprovedbytheFrenchMinistryofScienceandTechnology
(agreementno.AC2009-921).Humanperipheralbloodmononuclearcellswere
establish that the alternative polarization of macrophages by
isolatedfromthebloodofhealthyvolunteersbyadensitygradientcentrifugation
IL-13 is dependent on the STAT6/LRH-1/CYPs/15-HETE/ methodonLymphoprep(Abcys).Monocyteswereisolatedbyadherencetoplastic
PPARg axis (Fig. 7). Finally, deletion of LRH-1 in myeloid cells for2hinSFM(Gibco)at37(cid:2)C,5%CO.Themacrophageswereobtainedafter
2
renders mice susceptible to gastrointestinal and systemic C. 3daysofcultureonlyinSFMmedium.
albicans infection, highlighting LRH-1 as a critical factor for
antifungal functions. Synthetic agonists of LRH-1 activity may, Preparationofmouseresidentperitonealmacrophages. Afterbeingkilled,
hence, constitute promising compounds for the treatment of residentperitonealcellswereharvestedbywashingtheperitonealcavitywith5ml
anti-infectious and anti-inflammatory diseases. ofsterileNaCl0.9%.Collectedcellswerecentrifugedat1,500r.p.m.for10minand
thecellpelletwassuspendedinDulbecco’smodifiedEagle’smedium(Invitrogen)
supplementedwithglutamine(Invitrogen),penicillin,streptomycin(Invitrogen)
and5%heat-inactivatedfetalcalfserum.Cellswereallowedtoadherefor2hat
Methods
37(cid:2)Cand5%CO.NonadherentcellswerethenremovedbywashingwithPBS.
Mice.Malemiceaged10–12weeksonC57BL/6backgroundwereusedforinvitro 2
andinvivoexperiments.Micewerebredandhandledbyfollowingprotocols
approvedbytheConseilScientifiqueduCentredeFormationetdeRecherche Reversetranscriptionandreal-timePCR.Afterwashing,adherentmacrophages
ExperimentalMe´dicoChirurgicalandtheethicsboardoftheMidi-Pyre´ne´esethic wereimmediatelystimulatedwithIFNg(40UIml(cid:2)1,Clinisciences),IL-6
committeeforanimalexperimentation(Experimentationpermitnumber31–067, (50ngml(cid:2)1,Clinisciences),LPS(1ngml(cid:2)1,Sigma),IL-4(50ngml(cid:2)1,Miltenyi
approvalno.B3155503).Allcageswerechangedtwiceweekly,andallmanipula- Biotech),IL-13(50ngml(cid:2)1,Clinisciences),IL-10(50ngml(cid:2)1,Clinisciences),
tionsoftheanimalswerecarriedoutinalaminalblowhoodunderasepticcon- 15-HETE(1mM,Cayman)orDLPC(50mM,Sigma)for4or24h.Inindicated
ditions.Thephotoperiodwasadjustedto12-hlightand12-hdark.C57BL/6mice experiments,adherentmacrophageswerepre-incubatedornotwithaJak-2/STAT6
werepurchasedfromJanvier(France)andStat6(cid:2)/(cid:2) miceandALox15(cid:2)/(cid:2) mice inhibitor,AG490(1nM,Tebu-Bio).
10 NATURECOMMUNICATIONS|6:6801|DOI:10.1038/ncomms7801|www.nature.com/naturecommunications
&2015MacmillanPublishersLimited.Allrightsreserved.